Coagulation factor VII polypeptides

ABSTRACT

The present invention relates to modified coagulation Factor VII polypeptides exhibiting increased resistance to antithrombin inactivation and enhanced proteolytic activity. The present invention also relates to polynucleotide constructs encoding such polypeptides, vectors and host cells comprising and expressing such polynucleotides, pharmaceutical compositions, uses and methods of treatment.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119 of U.S.Provisional Application 61/895,438, filed Oct. 25, 2013; thisapplication further claims priority of European Application 13188715.0,filed Oct. 15, 2013, and European Application 14154875.0, filed Feb. 12,2014; the contents of all above-named applications are incorporatedherein by reference.

TECHNICAL FIELD

The present invention relates to coagulation Factor VII (Factor VII)polypeptides having pro-coagulant activity. It also relates topharmaceutical compositions comprising such polypeptides, methods oftreatment and uses of such polypeptides.

SEQUENCE LISTING

SEQ ID NO. 1: Wild type human coagulation Factor VII.

SEQ ID NO. 2: Protease domain of human coagulation Factor VII.

SEQ ID NO. 3: Protease domain of hominin (chimpanzee) coagulation FactorVII.

SEQ ID NO. 4: Protease domain of canine (dog) coagulation Factor VII.

SEQ ID NO. 5: Protease domain of porcine (pig) coagulation Factor VII.

SEQ ID NO. 6: Protease domain of bovine (cattle) coagulation Factor VII.

SEQ ID NO. 7: Protease domain of murine (mouse) coagulation Factor VII.

SEQ ID NO. 8: Protease domain of murine (rat) coagulation Factor VII.

SEQ ID NO. 9: Protease domain of lapine (rabbit) coagulation Factor VII.

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 14, 2014, isnamed 8719US02_SeqListing_ST25 and is 22 kilobytes in size.

BACKGROUND OF INVENTION

An injury to a blood vessel activates the haemostatic system thatinvolves complex interactions between cellular and molecular components.The process that eventually causes the bleeding to stop is known ashaemostasis. An important part of haemostasis is coagulation of theblood and the formation of a clot at the site of the injury. Thecoagulation process is highly dependent on the function of severalprotein molecules. These are known as coagulation factors. Some of thecoagulation factors are proteases which can exist in an inactive zymogenor an enzymatically active form. The zymogen form can be converted toits enzymatically active form by specific cleavage of the polypeptidechain catalyzed by another proteolytically active coagulation factor.

Factor VII is a vitamin K-dependent plasma protein synthesized in theliver and secreted into the blood as a single-chain glycoprotein. TheFactor VII zymogen is converted into an activated form (Factor VIIa) byspecific proteolytic cleavage at a single site, i.e. between R152 and1153 of SEQ ID NO: 1, resulting in a two chain molecule linked by asingle disulfide bond. The two polypeptide chains in Factor VIIa arereferred to as light and heavy chain, corresponding to residues 1-152and 153-406, respectively, of SEQ ID NO: 1 (wild type human coagulationFactor VII). Factor VII circulates predominantly as zymogen, but a minorfraction is in the activated form (Factor VIIa).

The blood coagulation process can be divided into three phases:initiation, amplification and propagation. The initiation andpropagation phases contribute to the formation of thrombin, acoagulation factor with many important functions in haemostasis. Thecoagulation cascade starts if the single-layered barrier of endothelialcells that line the inner surface of blood vessels becomes damaged. Thisexposes subendothelial cells and extravascular matrix proteins to whichplatelets in the blood will stick to. If this happens, Tissue Factor(TF) which is present on the surface of sub-endothelial cells becomesexposed to Factor VIIa circulating in the blood. TF is a membrane-boundprotein and serves as the receptor for Factor VIIa. Factor VIIa is anenzyme, a serine protease, with intrinsically low activity. However,when Factor VIIa is bound to TF, its activity increases greatly. FactorVIIa interaction with TF also localizes Factor VIIa on the phospholipidsurface of the TF bearing cell and positions it optimally for activationof Factor X to Xa. When this happens, Factor Xa can combine with FactorVa to form the so-called “prothombinase” complex on the surface of theTF bearing cell. The prothrombinase complex then generates thrombin bycleavage of prothrombin. The pathway activated by exposing TF tocirculating Factor VIIa and leading to the initial generation ofthrombin is known as the TF pathway. The TF:Factor VIIa complex alsocatalyzes the activation of Factor IX to Factor IXa. Then activatedFactor IXa can diffuse to the surface of platelets which are sticking tothe site of the injury and have been activated. This allows Factor IXato combine with FVIIIa to form the “tenase” complex on the surface ofthe activated platelet. This complex plays a key role in the propagationphase due to its remarkable efficiency in activating Factor X to Xa. Theefficient tenase catalyzed generation of Factor Xa activity in turnleads to efficient cleavage of prothrombin to thrombin catalyzed by theprothrombinase complex.

If there are any deficiencies in either Factor IX or Factor VIII, itcompromises the important tenase activity, and reduces the production ofthe thrombin which is necessary for coagulation. Thrombin formedinitially by the TF pathway serves as a pro-coagulant signal thatencourages recruitment, activation and aggregation of platelets at theinjury site. This results in the formation of a loose primary plug ofplatelets. However, this primary plug of platelets is unstable and needsreinforcement to sustain haemostasis. Stabilization of the plug involvesanchoring and entangling the platelets in a web of fibrin fibres.

The formation of a strong and stable clot is dependent on the generationof a robust burst of local thrombin activity. Thus, deficiencies in theprocesses leading to thrombin generation following a vessel injury canlead to bleeding disorders e.g. haemophilia A and B. People withhaemophilia A and B lack functional Factor Villa or Factor IXa,respectively. Thrombin generation in the propagation phase is criticallydependent on tenase activity, i.e. requires both Factor Villa and FIXa.Therefore, in people with haemophilia A or B proper consolidation of theprimary platelet plug fails and bleeding continues.

Replacement therapy is the traditional treatment for hemophilia A and B,and involves intravenous administration of Factor VIII or Factor IX. Inmany cases, however, patients develop antibodies (also known asinhibitors) against the infused proteins, which reduce or negate theefficacy of the treatment. Recombinant Factor VIIa (Novoseven®) has beenapproved for the treatment of hemophilia A or B patients withinhibitors, and also is used to stop bleeding episodes or preventbleeding associated with trauma and/or surgery. Recombinant Factor VIIahas also been approved for the treatment of patients with congenitalFactor VII deficiency. It has been proposed that recombinant FVIIaoperates through a TF-independent mechanism. According to this model,recombinant FVIIa is directed to the surface of the activated bloodplatelets by virtue of its Gla-domain where it then proteolyticallyactivates Factor X to Xa thus by-passing the need for a functionaltenase complex. The low enzymatic activity of FVIIa in the absence of TFas well as the low affinity of the Gla-domain for membranes couldexplain the need for supra-physiological levels of circulating FVIIaneeded to achieve haemostasis in people with haemophilia.

Recombinant Factor VIIa has an in vivo functional half-life of 2-3 hourswhich may necessitate frequent administration to resolve bleedings inpatients. Further, patients often only receive Factor VIIa therapy aftera bleed has commenced, rather than as a precautionary measure, whichoften impinges upon their general quality of life. A recombinant FactorVIIa variant with a longer in vivo functional half-life would decreasethe number of necessary administrations, support less frequent dosingand thus holds the promise of significantly improving Factor VIIatherapy to the benefit of patients and care-holders.

WO02/22776 discloses Factor VIIa variants with enhanced proteolyticactivity compared to wild-type FVIIa. It has been demonstrated inclinical trials that a Factor VII polypeptide comprising substitutionsdisclosed in WO02/22776 shows a favourable clinical outcome in terms ofefficacy of a variant with enhanced proteolytic activity (de Paula et al(2012) J Thromb Haemost, 10:81-89).

WO2007/031559 discloses Factor VII variants with reduced susceptibilityto inhibition by antithrombin.

WO2009/126307 discloses modified Factor VII polypeptides with alteredprocoagulant activity.

In general, there are many unmet medical needs in people withcoagulopathies. The use of recombinant Factor VIIa to promote clotformation underlines its growing importance as a therapeutic agent.However, recombinant Factor VIIa therapy still leaves significant unmetmedical needs, a recombinant Factor VIIa polypeptides having improvedpharmaceutical properties, for example increased in vivo functionalhalf-life and enhanced or higher activity, would meet some of theseneeds.

SUMMARY OF THE INVENTION

The present invention provides Factor VII polypeptides that are designedto have improved pharmaceutical properties. In one broad aspect, theinvention relates to Factor VII polypeptides exhibiting increased invivo functional half-life as compared to human wild-type Factor VIIa. Inanother broad aspect, the invention relates to Factor VII polypeptideswith enhanced activity as compared to human wild-type Factor VIIa. In afurther broad aspect, the invention relates to Factor VII polypeptidesexhibiting increased resistance to inactivation by endogenous plasmainhibitors, particularly antithrombin, as compared to human wild-typeFactor VIIa.

Provided herein are Factor VII polypeptides with increased in vivofunctional half-life which comprise a combination of mutationsconferring resistance to antithrombin inactivation and enhanced orlittle or no loss of proteolytic activity. In a particularly interestingaspect of the present invention the Factor VII polypeptides are coupledto one or more “half-life extending moieties” to increase the in vivofunctional half-life.

In one aspect, the invention relates to a Factor VII polypeptidecomprising two or more substitutions relative to the amino acid sequenceof human Factor VII (SEQ ID NO:1), wherein T293 is replaced by Lys (K),Arg (R), Tyr (Y) or Phe (F) and L288 is replaced by Phe (F), Tyr (Y),Asn (N), or Ala (A) and/or W201 is replaced by Arg (R), Met (M) or Lys(K) and/or K337 is replaced by Ala (A) or Gly (G).

The Factor VII polypeptide of the invention may comprise a substitutionof T293 with Lys (K) and a substitution of L288 with Phe (F). The FactorVII polypeptide may comprise a substitution of T293 with Lys (K) and asubstitution of L288 with Tyr (Y). The Factor VII polypeptide maycomprise a substitution of T293 with Arg (R) and a substitution of L288with Phe (F). The Factor VII polypeptide may comprise a substitution ofT293 with Arg (R) and a substitution of L288 with Tyr (Y). The FactorVII polypeptide may comprise, or may further comprise, a substitution ofK337 with Ala (A). The Factor VII polypeptide may comprise asubstitution of T293 with Lys (K) and a substitution of W201 with Arg(R).

In an interesting embodiment the invention relates to Factor VIIpolypeptides coupled with at least one half-life extending moiety.

In another aspect, the invention relates to methods for producing theFactor VII polypeptides of the invention.

In a further aspect, the invention relates to pharmaceuticalcompositions comprising a Factor VII polypeptide of the invention.

The general object of the present invention is to improve currentlyavailable treatment options in people with coagulopathies and to obtainFactor VII polypeptides with improved therapeutic utility.

One object that the present invention has is to obtain Factor VIIpolypeptides with prolonged in vivo functional half-life whilemaintaining a pharmaceutically acceptable proteolytic activity. Toachieve this, the Factor VII polypeptides of the present inventioncomprise a combination of mutations conferring reduced susceptibility toinactivation by the plasma inhibitor antithrombin while substantiallypreserving proteolytic activity; in particularly interesting embodimentsof the present invention the Factor VII polypeptides are also coupled toone or more “half-life extending moieties”.

Medical treatment with the modified Factor VII polypeptides of thepresent invention offers a number of advantages over the currentlyavailable treatment regimes, such as longer duration between injections,lower dosage, more convenient administration, and potentially improvedhaemostatic protection between injections.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows amino acid sequence alignment of the FVIIa protease domainfrom different species.

FIG. 2 shows the correlation between in vitro antithrombin reactivitywith the in vivo accumulation of FVIIa-antithrombin complexes.

FIG. 3 shows the conformation of arginine at position 201 in the FVIIavariant W201R T293Y double mutant compared to the conformation oftryptophan at position 201 in FVIIa WT.

FIG. 4 shows a hypothetical model of interaction between tyrosine atposition 293 from the FVIIa variant W201R T293Y double mutant with theantithrombin. This is based on a theoretical model of a complex betweenantithrombin and the FVIIa variant W201R T293Y double mutant shown instick representation. Antithrombin amino acids are depicted with aprefix “AT”; while, the FVIIa amino acids are depicted with a prefix“FVIIa”.

FIG. 5A shows structure of heparosan.

FIG. 5B shows structure of a heparosan polymer with maleimidefunctionality at its reducing end.

FIG. 6A and FIG. 6B show assessment of conjugate purity by SDS-PAGE.FIG. 6A shows SDS-PAGE analysis of final FVIIa conjugates. Gel wasloaded with HiMark HMW standard (lane 1); FVIIa (lane 2);13k-HEP-[C]-FVIIa (lane 3); 27k-HEP-[C]-FVIIa (lane 4);40k-HEP-[C]-FVIIa (lane 5); 52k-HEP-[C]-FVIIa (lane 6);60k-HEP-[C]-FVIIa (lane 7); 65k-HEP-[C]-FVIIa (lane 8);108k-HEP-[C]-FVIIa (lane 9) and 157k-HEP-[C]FVIIa407C (lane 10). FIG. 6Bshows SDS-PAGE of glycoconjugated 52k-HEP-[N]-FVIIa. Gel was loaded withHiMark HMW standard (lane 1), ST3Gal3 (lane 2), FVIIa (lane 3), asialoFVIIa (lane 4), and 52k-HEP-[N]-FVIIa (lane 5). [N]-denotes Factorconjugates where HEParosan is attached to the N-glycan. [C]-DenotesFactor conjugates where Heparosan is attached to a cystein residue.

FIG. 7: Analysis of FVIIa clotting activity levels of heparosanconjugates and glycoPEGylated FVIIa references.

FIG. 8: Proteolytic activity of heparosan conjugates and glycoPEGylatedFVIIa references.

FIG. 9: PK results (LOCI) in Sprague Dawley rats. Comparison ofunmodified FVIIa (2 studies), 13k-HEP-[C]FVIIa407C,27k-HEP-[C]FVIIa407C, 40k-HEP-[C]-FVIIa407C, 52k-HEP-[C]FVIIa407C,65k-HEP-[C]FVIIa407C, 108k-HEP-[C]FVIIa407C and 157k-HEP-[C]FVIIa407C,glycoconjugated 52k-HEP-[N]-FVIIa and reference molecules (40kDa-PEG-[N]FVIIa (2 studies) and 40 kDa-PEG-[C]FVIIa407C). Data areshown as mean±SD (n=3-6) in a semilogarithmic plot. [N]-denotes Factorconjugates where HEParosan is attached to the N-glycan. [C]-DenotesFactor conjugates where Heparosan is attached to a cystein residue.

FIG. 10: PK results (Clot Activity) in Sprague Dawley rats. Comparisonof unmodified FVIIa (2 studies), 13k-HEP-[C]FVIIa407C,27k-HEP-[C]-FVIIa407C, 40k-HEP-[C]-FVIIa407C, 52k-HEP-[C]FVIIa407C,65k-HEP-[C]FVIIa407C, 108k-HEP-[C]FVIIa407C and 157k-HEP-[C]FVIIa407C,glycoconjugated 52k-HEP-[N]-FVIIa and reference molecules (40kDa-PEG-[N]FVIIa (2 studies) and 40 kDa-PEG-[C]FVIIa407C). Data areshown in a semilogarithmic plot. [N]-denotes Factor conjugates whereHEParosan is attached to the N-glycan. [C]-Denotes Factor conjugateswhere Heparosan is attached to a cystein residue.

FIG. 11: Relationship between HEP-size and mean residence time (MRT) fora number of HEP-[C]FVIIa407C conjugates. MRT values from PK studies areplotted against heparosan polymer size of conjugates. The plot representvalues for non-conjugated FVIIa, 13k-HEP-[C]FVIIa407C,27k-HEP-[C]FVIIa407C, 40k-HEP-[C]FVIIa407C, 52k-HEP-[C]-FVIIa407C,65k-HEP-[C]FVIIa407C, 108k-HEP-[C]FVIIa407C and 157k-HEP-[C]-FVIIa407C.MRT (LOCI) was calculated by non-compartmental methods using PhoenixWinNonlin 6.0 (Pharsight Corporation). [N]-denotes Factor conjugateswhere HEParosan is attached to the N-glycan. [C]-Denotes Factorconjugates where Heparosan is attached to a cystein residue.

FIG. 12 Functionalization of glycylsialic acid cytidine monophosphate(GSC) with a benzaldehyde group. GSC is acylated with 4-formylbenzoicacid and subsequently reacted with heparosan (HEP)-amine by a reductiveaminination reaction.

FIG. 13: Functionalization of heparosan (HEP) polymer with abenzaldehyde group and subsequent reaction with glycylsialic acidcytidine monophosphate (GSC) in a reductive amination reaction.

FIG. 14: Functionalization of glycylsialic acid cytidine monophosphate(GSC) with a thio group and subsequent reaction with a maleimidefunctionalized heparosan (HEP) polymer.

FIG. 15: Heparosan (HEP)—glycylsialic acid cytidine monophosphate (GSC).

FIG. 16: PK results (LOCI) in Sprague Dawley rats. Comparison of2×20K-HEP-[N]-FVIIa; 1×40K-HEP-[N]FVIIa and 1×40k-PEG-[N]FVIIa in asemilogarithmic plot. Data are shown as mean±SD (n=3-6).

FIG. 17: PK results (Clot Activity) in Sprague Dawley rats. Comparisonof 2×20K-HEP-[N]-FVIIa; 1×40K-HEP-[N]FVIIa and 1×40k-PEG-[N]FVIIa in asemilogarithmic plot.

FIG. 18: Reaction scheme wherein an asialoFVIIa glycoprotein is reactedwith HEP-GSC in the presence of a ST3GalIII sialyltransferase.

DETAILED DESCRIPTION

The present invention relates to the design and use of Factor VIIpolypeptides exhibiting improved pharmaceutical properties.

In one aspect, the present invention relates to the design and use ofFactor VII polypeptides exhibiting increased in vivo functionalhalf-life, reduced susceptibility to inactivation by the plasmainhibitor antithrombin and enhanced or preserved proteolytic activity.It has been found by the inventors of the present invention thatspecific combinations of mutations in human Factor VII in combinationwith conjugation to half-life extending moieties confer the abovementioned properties. The Factor VII polypeptides of the invention havean extended functional half-life in blood which is therapeuticallyuseful in situations where a longer lasting pro-coagulant activity iswanted. Such Factor VII polypeptides comprise a substitution of T293with Lys (K), Arg (R), Tyr (Y) or Phe (F). In this aspect, the inventionrelates to a Factor VII polypeptide comprising two or more substitutionsrelative to the amino acid sequence of human Factor VII (SEQ ID NO:1),wherein T293 is replaced by Lys (K), Arg (R), Tyr (Y) or Phe (F) andL288 is replaced by Phe (F), Tyr (Y), Asn (N), Ala (A), or Trp (W). Theinvention also relates to a Factor VII polypeptide comprising two ormore substitutions relative to the amino acid sequence of human FactorVII (SEQ ID NO:1), wherein T293 is replaced by Lys (K), Arg (R), Tyr (Y)or Phe (F) and W201 is replaced by Arg (R), Met (M), or Lys (K).Furthermore, the invention relates to a Factor VII polypeptidecomprising two or more substitutions relative to the amino acid sequenceof human Factor VII (SEQ ID NO:1), wherein T293 is replaced by Lys (K),Arg (R), Tyr (Y) or Phe (F) and K337 is replaced by Ala (A) or Gly (G).Optionally, Factor VII polypeptides of the invention may furthercomprise substitution of Q176 with Lys (K), Arg (R) or Asn (N).Optionally, Factor VII polypeptides of the invention may furthercomprise substitution of Q286 with Asn (N).

In another aspect, the present invention relates to the design and useof Factor VII polypeptides exhibiting enhanced proteolytic activity. Ithas been found by the inventors of the present invention that specificmutations in human Factor VII at positions L288 and/or W201 conferenhanced proteolytic activity to Factor VII polypeptides. In thisaspect, the invention relates to a Factor VII polypeptide comprising oneor more substitutions relative to the amino acid sequence of humanFactor VII (SEQ ID NO: 1), wherein L288 is replaced by Phe (F), Tyr (Y),Asn (N), Ala (A) or Trp (W), with the proviso that the polypeptide doesnot have the following pair of substitutions: L288N/R290S orL288N/R290T. Further according to this aspect, the invention relates toa Factor VII polypeptide comprising one or more substitutions relativeto the amino acid sequence of human Factor VII (SEQ ID NO:1),characterized in that one substitution is where W201 is replaced by Arg(R), Met (M) or Lys (K).

Factor VII

Coagulation Factor VII (Factor VII) is a glycoprotein primarily producedin the liver. The mature protein consists of 406 amino acid residuesdefined by SEQ ID NO: 1 (also disclosed in, for example, in U.S. Pat.No. 4,784,950) and is composed of four domains. There is an N-terminalgamma-carboxyglutamic acid (Gla) rich domain followed by two epidermalgrowth factor (EGF)-like domains and a C-terminal trypsin-like serineprotease domain. Factor VII circulates in plasma, predominantly as asingle-chain molecule. Factor VII is activated to Factor VIIa bycleavage between residues Arg152 and Ile153, resulting in a two-chainprotein held together by a disulphide bond. The light chain contains theGla and EGF-like domains, while the heavy chain is the protease domain.Specific Glu (E) residues, i.e. E6, E7, E14, E16, E19, E20, E25, E26,E29 and E35, according to SEQ ID NO: 1 in Factor VII may bepost-translationally gamma-carboxylated. The gamma-carboxyglutamic acidresidues in the Gla domain are required for coordination of a number ofcalcium ions, which maintain the Gla domain in a conformation mediatinginteraction with phospholipid membranes.

The terms FVII and “Factor VII” herein refers to the uncleavedsingle-chain zymogen, Factor VII, as well as the cleaved, two-chain andthus activated protease, Factor VIIa. “Factor VII” includes naturalallelic variants of Factor VII that may exist and differ from oneindividual to another. A human wild-type Factor VII sequence is providedin SEQ ID NO: 1. The term “Factor VII polypeptide” herein refers to theuncleaved single chain zymogen polypeptide variant of Factor VII (asdescribed herein), as well as the cleaved, two chain and thus activatedprotease.

Factor VII and Factor VII polypeptides may be plasma-derived orrecombinantly produced, using well known methods of production andpurification. The degree and location of glycosylation,gamma-carboxylation and other post-translational modifications may varydepending on the chosen host cell and its growth conditions.

Factor VII Polypeptides

The terms “Factor VII” or “FVII” denote Factor VII polypeptides.

The term “Factor VII polypeptide” encompasses wild type Factor VIImolecules as well as Factor VII variants, Factor VII conjugates andFactor VII that has been chemically modified. Such, variants, conjugatesand chemically modified Factor VII may exhibit substantially the same,or improved, activity relative to wild-type human Factor VIIa.

The term “activity” of a Factor VII polypeptide, as used herein, refersto any activity exhibited by wild-type human Factor VII, and includes,but is not limited to, coagulation or coagulant activity, pro-coagulantactivity, proteolytic or catalytic activity such as to effect Factor Xactivation or Factor IX activation; ability to bind TF, Factor X orFactor IX; and/or ability to bind to phospholipids. These activities canbe assessed in vitro or in vivo using recognized assays, for example, bymeasuring coagulation in vitro or in vivo. The results of such assaysindicate that a polypeptide exhibits an activity that can be correlatedto activity of the polypeptide in vivo, in which in vivo activity can bereferred to as biological activity. Assays to determine activity of aFactor VII polypeptide are known to those of skill in the art. Exemplaryassays to assess the activity of a FVII polypeptide include in vitroproteolysis assays, such as those described in the Examples, below.

The terms “enhanced, or preserved activity”, as used herein, refer toFactor VIIa polypeptides that exhibit substantially the same orincreased activity compared to wild type human Factor VIIa, for examplei) substantially the same or increased proteolytic activity compared torecombinant wild type human Factor VIIa in the presence and/or absenceof TF; ii) to Factor VII polypeptides with substantially the same orincreased TF affinity compared to recombinant wild type human FactorVIIa; iii) to Factor VII polypeptides with substantially the same orincreased affinity for the activated platelet; or iv) Factor VIIpolypeptides with substantially the same or increased affinity/abilityto bind to Factor X or Factor IX compared to recombinant wild type humanFactor VIIa. For example preserved activity means that the amount ofactivity that is retained is or is about 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 100% or more of the activity compared to wild type humanFactor VIIa. For example enhanced activity means that the amount ofactivity that is retained is or is about 110%, 120%, 130%, 140%, 150%,160%, 170%, 180%, 190%, 200%, 300%, 400%, 500%, 1000%, 3000%, 5000%, 10000%, 30 000% or more of the activity compared to wild type human FactorVIIa.

The term “Factor VII variant”, as used herein, is intended to designatea Factor VII having the sequence of SEQ ID NO: 1, wherein one or moreamino acids of the parent protein have been substituted by anothernaturally occurring amino acid and/or wherein one or more amino acids ofthe parent protein have been deleted and/or wherein one or more aminoacids have been inserted in the protein and/or wherein one or more aminoacids have been added to the parent protein. Such addition can takeplace either at the N- or at the C-terminus of the parent protein orboth. In one embodiment a variant is at least 95% identical with thesequence of SEQ ID NO: 1. In another embodiment a variant is at least80, 85, 90, 95, 96, 97, 98 or 99% identical with the sequence of SEQ IDNO: 1. As used herein, any reference to a specific position refers tothe corresponding position in SEQ ID NO: 1.

In some embodiments, the Factor VII variants of this invention have anenhanced or preserved activity compared to wild type human Factor VIIa.

The terminology for amino acid substitutions used in this description isas follows. The first letter represents the amino acid naturally presentat a position of SEQ ID NO:1. The following number represent theposition in SEQ ID NO:1. The second letter represents the differentamino acid substituting the natural amino acid. An example isK197A-Factor VII, wherein the Lysine at position 197 of SEQ ID NO:1 isreplaced by a Alanine.

In the present context the three-letter or one-letter abbreviations ofthe amino acids have been used in their conventional meaning asindicated in below. Unless indicated explicitly, the amino acidsmentioned herein are L-amino acids.

Abbreviations for Amino Acids:

Amino acid Three-letter code One-letter code Glycine Gly G Proline Pro PAlanine Ala A Valine Val V Leucine Leu L Isoleucine Ile I Methionine MetM Cysteine Cys C Phenylalanine Phe F Tyrosine Tyr Y Tryptophan Trp WHistidine His H Lysine Lys K Arginine Arg R Glutamine Gln Q AsparagineAsn N Glutamic Acid Glu E Aspartic Acid Asp D Serine Ser S Threonine ThrT

The term “Factor VII conjugates” as used herein, is intended todesignate a Factor VII polypeptide, in which one or more of the aminoacids and/or one or more of the attached glycan moieties have beenchemically and/or enzymatically modified, such as by alkylation,glycosylation, acylation, ester formation, disulfide bond formation, oramide formation.

In some embodiments, the Factor VII conjugates of the invention exhibitsubstantially the same or enhanced biological activity relative towild-type Factor VII.

Enhanced Proteolytic Activity

Factor VII polypeptides with certain mutations of residues L288 and W201have, surprisingly, been shown by the inventors to exhibit enhancedproteolytic activity.

The Factor VII variant K337A, as described in WO02/22776, has beendescribed to have enhanced proteolytic activity. The Factor VII variantsL305V and L305I, as described in WO03/027147, have also been describedto have higher intrinsic activity.

The proteolytic activity may be determined by any suitable method knownin the art as further discussed below.

For example enhanced proteolytic activity means that the amount ofactivity that is retained is or is about 110%, 120%, 130%, 140%, 150%,160%, 170%, 180%, 190%, 200%, 300%, 400%, 500%, 1000%, 3000%, 5000%, 10000%, 30 000% or more of the activity compared to wild type human FactorVIIa.

Half-Life—Resistance to Inactivation by Plasma Inhibitors

Besides in vivo clearance, in vivo functional half-life is of importanceto the period of time during which the compound is “therapeuticallyavailable” in the body. The circulating half-life of recombinant humanwild type Factor VIIa is about 2.3 hours (“Summary Basis for Approvalfor NovoSeven©”, FDA reference number 96 0597).

The term “in vivo functional half-life” is used in its normal meaning,i.e., the time required for reducing the biological activity of theFactor VII polypeptide remaining in the body/target organ with 50% inthe terminal phase, or the time at which the activity of the Factor VIIpolypeptide is 50% of its initial value. Alternative terms to in vivohalf-life include terminal half-life, plasma half-life, circulatinghalf-life, circulatory half-life, and clearance half-life. Half-life maybe determined by suitable methods known in the art, such as thatdescribed in Example 17 and those described in Introduction toPharmacokinetics and Pharmacodynamics: The Quantitative Basis of DrugTherapy (Thomas N. Tozer, Malcolm Rowland).

The term “increased” as used about the in vivo functional half-life orplasma half-life is used to indicate that the relevant half-life of thepolypeptide is increased relative to that of a reference molecule, suchas wild-type human Factor VIIa as determined under comparableconditions.

In some embodiments, the Factor VII polypeptides of the inventionexhibit increased in vivo functional half-life relative to wild-typehuman Factor VIIa. For instance the relevant half-life may be increasedby at least about 25%, such as by at least about 50%, e.g., by at leastabout 100%, 150%, 200%, 500%, 1000%, 3000%, 5000%, 10 000%, 30 000% ormore.

Despite the detailed understanding of the biochemistry andpathophysiology of the coagulation cascade, the mechanistic basis forthe clearance of the individual coagulation factors from circulationremains largely unknown. The marked differences in the circulatinghalf-lives of Factor VII and its activated form Factor VIIa comparedwith zymogen and enzyme forms of other vitamin K-dependent proteinssuggest the existence of specific and distinct clearance mechanisms forFactor VIIa. Two types of pathways appear to be operable in theclearance of Factor VIIa—one resulting in elimination of the intactprotein, the other mediated by plasma inhibitors and leading toproteolytic inactivation.

Antithrombin III (antithrombin, AT) is an abundant plasma inhibitor andtargets most proteases of the coagulation system, including Factor VIIa.It is present at micromolar concentrations in plasma and belongs to theserpin family of serine protease inhibitors that irreversibly bind andinactivate target proteases by a suicide substrate mechanism. Theinhibition by antithrombin appears to constitute the predominantclearance pathway of recombinant Factor VIIa in vivo followingintravenous administration. In a recent study of the pharmacokinetics ofrecombinant Factor VIIa in haemophilia patients, about 60% of the totalclearance could be attributed to this pathway (Agerso et al. (2011) JThromb Haemost, 9, 333-338).

In some embodiments, the Factor VII polypeptides of the inventionexhibit increased resistance to inactivation by the endogenous plasmainhibitors, particularly antithrombin, relative to wild-type humanFactor VIIa.

It has been found by the inventors of the present invention that bycombining the two groups of mutations mentioned above, i.e. mutationsconferring increased AT resistance and mutations conferring enhancedproteolytic activity, an increased or preserved activity is achievedwhile maintaining high resistance to inhibitor inactivation. That is,the Factor VII polypeptides of the present invention comprising acombination of mutations exhibit increased resistance to antithrombininactivation as well as substantially preserved proteolytic activity.When the Factor VII polypeptides of the invention are conjugated withone or more half-life extending moieties a surprisingly improved effecton half-life extension is achieved. Given these properties, suchconjugated Factor VII polypeptides of the invention exhibit increasedcirculatory half-life while maintaining a pharmaceutically acceptableproteolytic activity. Consequently, a lower dose of such conjugatedFactor VII polypeptide may be required to obtain a functionally adequateconcentration at the site of action and thus it will be possible toadminister a lower dose and/or with lower frequency to the subjecthaving bleeding episodes or needing enhancement of the normalhaemostatic system.

Additional Modifications

The Factor VII polypeptides of the invention may comprise furthermodifications, in particular further modifications which conferadditional advantageous properties to the Factor VII polypeptide. Thus,in addition to the amino acid substitutions mentioned above, the FactorVII polypeptides of the invention may for example comprise further aminoacid modification, e.g. one further amino acid substitution. In one suchembodiment, the Factor VII polypeptide of the invention has anadditional mutation or addition selected from the group R396C, Q250C,and 407C, as described e.g. in WO2002077218.

The Factor VII polypeptides of the invention may comprise additionalmodifications that are or are not in the primary sequence of the FactorVII polypeptide. Additional modifications include, but are not limitedto, the addition of a carbohydrate moiety, the addition of a half-lifeextending moiety, e.g. the addition of a, PEG moiety, an Fc domain, etc.For example, such additional modifications can be made to increase thestability or half-life of the Factor VII polypeptide.

Half-Life Extending Moieties or Groups

The term “half-life extending moieties” are herein used interchangeablyand understood to refer to one or more chemical groups attached to oneor more amino acid site chain functionalities such as —SH, —OH, —COOH,—CONH2, —NH2, and/or one or more N- and/or O-glycan structures and thatcan increase in vivo functional half-life of proteins/polypeptides whenconjugated/coupled to these proteins/polypeptides.

The in vivo functional half-life may be determined by any suitablemethod known in the art as further discussed below (Example 17).

Examples of half-life extending moieties include: Biocompatible fattyacids and derivatives thereof, Hydroxy Alkyl Starch (HAS) e.g. HydroxyEthyl Starch (HES), Poly Ethylen Glycol (PEG), Poly (Glyx-Sery)n (HAP),Hyaluronic acid (HA), Heparosan polymers (HEP), Phosphorylcholine-basedpolymers (PC polymer), Fleximers, Dextran, Poly-sialic acids (PSA), Fcdomains, Transferrin, Albumin, Elastin like (ELP) peptides, XTENpolymers, PAS polymers, PA polymers, Albumin binding peptides, CTPpeptides, FcRn binding peptides and any combination thereof.

In a particularly interesting embodiment, the Factor VII polypeptide ofthe invention is coupled with one or more half-life extending moieties.

In one embodiment, a cysteine-conjugated Factor VII polypeptide of theinvention have one or more hydrophobic half-life extending moietiesconjugated to a sulfhydryl group of a cysteine introduced in the FactorVII polypeptide. It is furthermore possible to link half-life extendingmoieties to other amino acid residues.

In one embodiment, the Factor VII polypeptide of the invention isdisulfide linked to tissue factor, as described e.g. in WO2007115953.

In another embodiment, the Factor VII polypeptide of the invention is aFactor VIIa variant with increased platelet affinity.

Heparosan Conjugates

Factor VII polypeptide heparosan conjugates according to the presentinvention may have one or more Heparosan polymer (HEP) moleculesattached to any part of the FVII polypeptide including any amino acidresidue or carbohydrate moiety of the Factor VII polypeptide. Examplesof such conjugates are provided in WO2014/060397, which is hereinincorporated by reference. Chemical and/or enzymatic methods can beemployed for conjugating HEP to a glycan on the Factor VII polypeptide.An example of an enzymatic conjugation process is described e.g. inWO03031464. The glycan may be naturally occurring or it may beengineered in, e.g. by introduction of an N-glycosylation motif (NXT/Swhere X is any naturally occurring amino acid) in the amino acidsequence of Factor VII using methods well known in the art.

“Cysteine-HEP Factor VII polypeptide conjugates” according to thepresent invention have one or more HEP molecules conjugated to asulfhydryl group of a cysteine residue present or introduced in the FVIIpolypeptide.

In one interesting embodiment of the invention, the Factor VIIpolypeptide is coupled to a HEP polymer. In one embodiment the HEPpolymer coupled to the Factor VII polypeptide has a molecular weight ina range selected from 13-65 kDa, 13-55 kDa, 25-55 kDa, 25-50 kDa, 25-45kDa, 30-45 kDa, 36-44 kDa and 38-42 kDa, or a molecular weight of 40kDa.

In one interesting embodiment of the invention, the Factor VIIpolypeptide is coupled to a HEP polymer on an N-glycan of the Factor VIIpolypeptide.

In a further embodiment of the invention, two HEP polymers are coupledto the same Factor VII polypeptide via N-glycans. In this embodimenteach of the HEP polymer coupled to the Factor VII polypeptide has amolecular weight in a range selected from 13-65 kDa, 13-55 kDa, 25-55kDa, 25-50 kDa, 25-45 kDa, 30-45 kDa, 36-44 kDa and 38-42 kDa, or amolecular weight of 40 kDa. Preferably, the polymers have identicalmolecular weight.

In a specific embodiment two 20 kDa-HEP polymers are coupled to the sameFactor VII polypeptide via its N-glycans.

In a specific embodiment two 30 kDa-HEP polymers are coupled to the sameFactor VII polypeptide via its N-glycans.

In a specific embodiment two 40 kDa-HEP polymers are coupled to the sameFactor VII polypeptide via its N-glycans.

Heparosan Polymers

Heparosan (HEP) is a natural sugar polymer comprising(-GlcUA-beta1,4-GlcNAc-alpha1,4-) repeats (see FIG. 5A). It belongs tothe glycosaminoglycan polysaccharide family and is a negatively chargedpolymer at physiological pH. It can be found in the capsule of certainbacteria but it is also found in higher vertebrates, where it serves asprecursor for the natural polymers heparin and heparan sulphate.Although not proven in detail, heparosan is believed to be degraded inthe lysosomes. An injection of a 100 kDa heparosan polymer labelled withBolton-Hunter reagents has shown that heparosan is secreted as smallerfragments in body fluids/waste (US 2010/0036001).

Heparosan polymers and methods of making such polymers are described inUS 2010/0036001, the content of which is incorporated herein byreference. In accordance with the present invention, the heparosanpolymer may be any heparosan polymer described or disclosed in US2010/0036001.

For use in the present invention, heparosan polymers can be produced byany suitable method, such as any of the methods described in US2010/0036001 or US 2008/0109236. Heparosan can be produced usingbacterial-derived enzymes. For example, the heparosan synthase PmHS1 ofPasteurella multocida Type D polymerises the heparosan sugar chain bytransferring both GlcUA and GlcNAc. The Escherichia coli K5 enzymes KfiA(alpha GlcNAc transferase) and KfiC (beta GlcUA transferase) cantogether also form the disaccharide repeat of heparosan.

A heparosan polymer for use in the present invention is typically apolymer of the formula (-GlcUA-beta1,4-GlcNAc-alpha1,4-)_(n).

The size of the heparosan polymer may be defined by the number ofrepeats n in this formula. The number of said repeats n may be, forexample, from 2 to about 5000. The number of repeats may be, for example50 to 2000 units, 100 to 1000 units or 200 to 700 units. The number ofrepeats may be 200 to 250 units, 500 to 550 units or 350 to 400 units.Any of the lower limits of these ranges may be combined with any higherupper limit of these ranges to form a suitable range of numbers of unitsin the heparosan polymer.

The size of the heparosan polymer may be defined by its molecularweight. The molecular weight may be the average molecular weight for apopulation of heparosan polymer molecules, such as the weight averagemolecular mass.

Molecular weight values as described herein in relation to size of theheparosan polymer may not, in practise, exactly be the size listed. Dueto batch to batch variation during heparosan polymer production, somevariation is to be expected. To encompass batch to batch variation, itis therefore to be understood, that a variation around +/− 10%, 9%, 8%,7%, 6%, 5%, 4%, 3%, 2% or 1% around target HEP polymer size should beexpected. For example HEP polymer size of 40 kDa denotes 40 kDa+/−10%,e.g. 40 kDa could for example in practise mean 38.8 kDa, 41.5 kDa or43.8 kDa

The heparosan polymer may have a molecular weight of, for example, 500Da to 1,000 kDa. The molecular weight of the polymer may be 500 Da to650 kDa, 5 kDa to 750 kDa, 10 kDa to 500 kDa, 15 kDa to 550 kDa or 25kDa to 250 kDa.

The molecular weight may be selected at particular levels within theseranges in order to achieve a suitable balance between activity of theFactor VII polypeptide and half-life or mean residence time of theconjugate. For example, the molecular weight of the polymer may be in arange selected from 15-25 kDa, 25-35 kDa, 35-45 kDa, 45-55 kDa, 55-65kDa or 65-75 kDa.

More specific ranges of molecular weight may be selected. For example,the molecular weight may be 20 kDa to 35 kDa, such as 22 kDa to 32 kDasuch as 25 kDa to 30 kDa, such as about 27 kDa. The molecular weight maybe 35 to 65 kDa, such as 40 kDa to 60 kDa, such as 47 kDa to 57 kDa,such as 50 kDa to 55 kDa such as about 52 kDa. The molecular weight maybe 50 to 75 kDa such as 60 to 70 kDa, such as 63 to 67 kDa such as about65 kDa.

In particularly interesting embodiments, the heparosan polymer of theFactor VII conjugate, of the invention, has a size in a range selectedfrom 13-65 kDa, 13-55 kDa, 25-55 kDa, 25-50 kDa, 25-45 kDa, 30-45 kDaand 38-42 kDa.

Any of the lower limits of these ranges of molecular weight may becombined with any higher upper limit from these ranges to form asuitable range for the molecular weight of the heparosan polymer inaccordance with the invention.

The heparosan polymer may have a narrow size distribution (i.e. bemonodisperse) or a broad size distribution (i.e. be polydisperse). Thelevel of polydispersity (PDI) may be represented numerically based onthe formula Mw/Mn, where Mw=weight average molecular mass and Mn=numberaverage molecular weight. The polydispersity value using this equationfor an ideal monodisperse polymer is 1. Preferably, a heparosan polymerfor use in the present invention is monodisperse. The polymer maytherefore have a polydispersity that is about 1, the polydispersity maybe less than 1.25, preferably less than 1.20, preferably less than 1.15,preferably less than 1.10, preferably less than 1.09, preferably lessthan 1.08, preferably less than 1.07, preferably less than 1.06,preferably less than 1.05.

The molecular weight size distribution of the heparosan may be measuredby comparison with monodisperse size standards (HA Lo-Ladder, HyaloseLLC) which may be run on agarose gels.

Alternatively, the size distribution of heparosan polymers may bedetermined by high performance size exclusion chromatography-multi anglelaser light scattering (SEC-MALLS). Such a method can be used to assessthe molecular weight and polydispersity of a heparosan polymer.

Polymer size may be regulated in enzymatic methods of production. Bycontrolling the molar ratio of heparosan acceptor chains to UDP sugar,it is possible to select a final heparosan polymer size that is desired

The heparosan polymer may further comprise a reactive group to allow itsattachment to a Factor VII polypeptide. A suitable reactive group maybe, for example, an aldehyde, alkyne, ketone, maleimide, thiol, azide,amino, hydrazide, hydroxylamine, carbonate ester, chelator or acombination of any thereof. For example, FIG. 5B illustrates a heparosanpolymer comprising a maleimide group.

As set out in the Examples, maleimide or aldehyde functionalizedheparosan polymers of defined size may be prepared by an enzymatic(PmHS1) polymerization reaction using the two sugar nucleotidesUDP-GlcNAc and UDP-GlcUA in equimolar amount. A priming trisaccharide(GlcUA-GlcNAc-GlcUA)NH₂ may be used for initiating the reaction, andpolymerization run until depletion of sugar nucleotide building blocks.Terminal amine (originating from the primer) may then be functionalizedwith suitable reactive groups such as a reactive group as describedabove, such as a maleimide functionality for conjugation to freecysteines or aldehydes for reductive amination to amino groups. The sizeof the heparosan polymers can be pre-determined by variation in sugarnucleotide: primer stoichiometry. The technique is described in detailin US 2010/0036001.

The reactive group may be present at the reducing or non-reducingtermini or throughout the sugar chain. The presence of only one suchreactive group is preferred when conjugating the heparosan polymer tothe polypeptide.

Methods for Preparing FVII-HEP Conjugates

For example, WO 03/031464 describes methods for remodelling the glycanstructure of a polypeptide, such as a Factor VII or Factor VIIapolypeptide and methods for the addition of a modifying group such as awater soluble polymer to such a polypeptide. Such methods may be used toattach a heparosan polymer to a Factor VII polypeptide in accordancewith the present invention.

As set out in the Examples, a Factor VII polypeptide may be conjugatedto its glycan moieties using sialyltransferase. For enablement of thisapproach, a HEP polymer first need to be linked to a sialic acidcytidine monophosphate. Glycylsialic acid cytidine monophosphate (GSC)is a suitable starting point for such chemistry, but other sialic acidcytidine monophosphate or fragments of such can be used. Examples setout methods for covalent linking HEP polymers to GSC molecules. Bycovalent attachment, a HEP-GSC (HEP conjugated glycylsialic acidcytidine monophosphate) molecule is created that can be transferred toglycan moieties of FVIIa.

Factor VII-heparosan conjugates may be purified once they have beenproduced. For example, purification may comprise affinity chromatographyusing immobilised mAb directed towards the Factor VII polypeptide, suchas mAb directed against the calcified gla-domain on FVIIa. In such anaffinity chromatography method, unconjugated HEP-polymer may be removedby extensive washing of the column. FVII may be released from the columnby releasing the FVII from the antibody. For example, where the antibodyis specific to the calcified gla-domain, release from the column may beachieved by washing with a buffer comprising EDTA.

Size exclusion chromatography may be used to separate FactorVII-heparosan conjugates from unconjugated Factor VII.

Pure conjugate may be concentrated by ultrafiltration.

Final concentrations of Factor VII-heparosan conjugate resulting from aprocess of production may be determined by, for example, HPLCquantification, such as HPLC quantification of the FVII light chain.

In connection with the present invention, it is shown that it ispossible to link a carbohydrate polymer such as HEP via a maleimidogroup to a thio-modified GSC molecule and transfer the reagent to anintact glycosyl group on a glycoprotein by means of a sialyltransferase,thereby creating a linkage that contains a cyclic succinimide group.

Succinimide based linkages, however, may undergo hydrolytic ring openingwhen the conjugate is stored in aqueous solution for extended timeperiods (Bioconjugation Techniques, G. T. Hermanson, Academic Press,3^(rd) edition 2013 p. 309) and while the linkage may remain intact, thering opening reaction will add undesirable heterogeneity in form ofregio- and stereo-isomers to the final conjugate.

It follows from the above that it is preferable to link the half-lifeextending moiety to the glycoprotein in such a way that 1) the glycanresidue of the glycoprotein is preserved in intact form, and 2) noheterogenicity is present in the linker part between the intact glycosylresidue and the half-life extending moiety.

There is a need in the art for methods of conjugating two compounds,such as a half-life extending moiety such as HEP to a protein or proteinglycan, wherein the compounds are linked such that a stable and isomerfree conjugate is obtained.

In one aspect the present invention provides a stable and isomer freelinker for use in glycylsialic acid cytidine monophosphate (GSC) basedconjugation of HEP to FVII. The GSC starting material used in thecurrent invention can be synthesised chemically (Dufner, G. Eur. J. Org.Chem. 2000, 1467-1482) or it can be obtained by chemoenzymatic routes asdescribed in WO07056191. The GSC structure is shown below:

In one embodiment conjugates according to the present invention comprisea linker comprising the following structure:

—hereinafter also referred to as sublinker or sublinkage—that connects aHEP-amine and GSC in one of the following ways:

The highlighted 4-methylbenzoyl sublinker thus makes up part of the fulllinking structure linking the half-life extending moiety to a targetprotein. The sublinker is as such a stable structure compared toalternatives, such as succinimide based linkers (prepared from maleimidereactions with sulfhydryl groups) since the latter type of cycliclinkage has a tendency to undergo hydrolytic ring opening when theconjugate is stored in aqueous solution for extended time periods(Bioconjugation Techniques, G. T. Hermanson, Academic Press, 3^(rd)edition 2013 p. 309). Even though the linkage in this case (e.g. betweenHEP and sialic acid on a glycoprotein) may remain intact, the ringopening reaction will add heterogeneity in form of regio- andstereo-isomers to the final conjugate composition.

One advantage associated with conjugates according to the presentinvention is thus that a homogenous composition is obtained, i.e. thatthe tendency of isomer formation due to linker structure and stabilityis significantly reduced. Another advantage is that the linker andconjugates according to the invention can be produced in a simpleprocess, preferably a one-step process.

Isomers are undesirable since these can lead to a heterogeneous productand increase the risk for unwanted immune responses in humans.

The 4-methylbenzoyl sublinkage as used in the present invention betweenHEP and GSC is not able to form sterio- or regio isomers. HEP polymerscan as mentioned earlier be prepared by a synchronised enzymaticpolymerisation reaction (US 20100036001). This method use heparansynthetase I from Pasturella multocida (PmHS1) which can be expressed inE. coli as a maltose binding protein fusion constructs. PurifiedMBP-PmHS1 is able to produce monodisperse polymers in a synchronized,stoichiometrically controlled reaction, when it is added to an equimolarmixture of sugar nucleotides (GlcNAc-UDP and GlcUA-UDP). A trisaccharideinitiator (GlcUA-GlcNAc-GlcUA) is used to prime the reaction, andpolymer length is determined by the primer:sugar nucleotide ratios. Thepolymerization reaction will run until about 90% of the sugarnucleotides are consumed. Polymers are isolated from the reactionmixture by anion exchange chromatography, and subsequently freeze-driedinto stable powder.

Processes for preparation of functional HEP polymers are described in US20100036001 which for example lists aldehyde-, amine- and maleimidefunctionalized HEP reagents. US 20100036001 is hereby incorporated byreference in its entirety as if fully set forth herein. A range of otherfunctionally modified HEP derivatives are available using similarchemistry. HEP polymers used in certain embodiments of the presentinvention are initially produced with a primary amine handle at thereducing terminal according to methods described in US20100036001.

Amine functionalized HEP polymers (i.e. HEP having an amine-handle)prepared according US20100036001 can be converted into aHEP-benzaldehyde by reaction with N-succinimidyl 4-formylbenzoate andsubsequently coupled to the glycylamino group of GSC by a reductiveamination reaction. The resulting HEP-GSC product can subsequently beenzymatically conjugated to a glycoprotein using a sialyltransferase.

For example, said amine handle on HEP can be converted into abenzaldehyde functionality by reaction with N-succinimidyl4-formylbenzoate according to the below scheme:

The conversion of HEP amine (1) to the 4-formylbenzamide compound (2) inthe above scheme may be carried out by reaction with acyl activatedforms of 4-formylbenzoic acid.

N-succinimidyl may be chosen as acyl activation group but a number ofother acyl activation groups are known to the skilled person.Non-limited examples include 1-hydroxy-7-azabenzotriazole-,1-hydroxy-benzotriazole-, pentafluorophenyl-esters as know from peptidechemistry.

HEP reagents modified with a benzaldehyde functionality can be keptstable for extended time periods when stored frozen (−80° C.) in dryform. Alternatively, a benzaldehyde moiety can be attached to the GSCcompound, thereby resulting in a GSC-benzaldehyde compound suitable forconjugation to an amine functionalized half-life extending moiety. Thisroute of synthesis is depicted in FIG. 12.

For example, GSC can be reacted under pH neutral conditions withN-succinimidyl 4-formylbenzoate to provide a GSC compound that containsa reactive aldehyde group (see for example WO2011101267). The aldehydederivatized GSC compound (GSC-benzaldehyde) can then be reacted withHEP-amine and reducing agent to form a HEP-GSC reagent.

The above mentioned reaction may be reversed, so that the HEP-amine isfirst reacted with N-succinimidyl 4-formylbenzoate to form an aldehydederivatized HEP-polymer, which subsequently is reacted directly with GSCin the presence of a reducing agent. In practice this eliminates thetedious chromatographic handling of GSC-CHO. This route of synthesis isdepicted in FIG. 13.

Thus, in one embodiment of the present invention HEP-benzaldehyde iscoupled to GSC by reductive amination.

Reductive amination is a two-step reaction which proceeds as follows:Initially an imine (also known as Schiff-base) is formed between thealdehyde component and the amine component (in the present embodimentthe glycyl amino group of GSC). The imine is then reduced to an amine inthe second step. The reducing agent is chosen so that it selectivelyreduces the formed imine to an amine derivative.

A number of suitable reducing reagents are available to the skilledperson. Non-limiting examples include sodium cyanoborohydride (NaBH3CN),sodium borohydride (NaBH4), pyridin boran complex (BH3:Py),dimethylsulfide boran complex (Me2S:BH3) and picoline boran complex.

Although reductive amination to the reducing end of carbohydrates (forexample to the reducing termini of HEP polymers) is possible, it hasgenerally been described as a slow and inefficient reaction (J C.Gildersleeve, Bioconjug Chem. 2008 July; 19(7): 1485-1490). Sidereactions, such as the Amadori reaction, where the initially formedimine rearrange to a keto amine are also possible, and will lead toheterogenicity which as previously discussed is undesirable in thepresent context.

Aromatic aldehydes such as benzaldehydes derivatives are not able toform such rearrangement reactions as the imine is unable to enolize andalso lack the required neighbouring hydroxy group typically found incarbohydrate derived imines. Aromatic aldehydes such as benzaldehydesderivatives are therefore particular useful in reductive aminationreactions for generating isomer free HEP-GSC reagent.

A surplus of GSC and reducing reagent is optionally used in order todrive reductive amination chemistry fast to completion. When thereaction is completed, the excess (non-reacted) GSC reagent and othersmall molecular components such as excess reducing reagent cansubsequently be removed by dialysis, tangential flow filtration or sizeexclusion chromatography.

Both the natural substrate for sialyltransferases, Sia-CMP, and the GSCderivatives are multifunctional molecules that are charged and highlyhydrophilic. In addition, they are not stable in solution for extendedtime periods especially if pH is below 6.0. At such low pH, the CMPactivation group necessary for substrate transfer is lost due to acidcatalyzed phosphate diester hydrolysis. Selective modification andisolation of GSC and Sia-CMP derivatives thus require careful control ofpH, as well as fast and efficient isolation methods, in order to avoidCMP-hydrolysis.

In the present invention, large half-life extending moieties areconjugated to GSC using reductive amination chemistry. Arylaldehydes,such as benzaldhyde modified half-life extending moieties have beenfound optimal for this type of modification, as they efficiently canreact with GSC under reductive amination conditions.

As GSC may undergo hydrolysis in acid media, it is important to maintaina near neutral or slightly basic environment during the coupling toHEP-benzaldehydes. HEP polymers and GSC are both highly water solubleand aqueous buffer systems are therefore preferable for maintaining pHat a near neutral level. A number of both organic and inorganic buffersmay be used, however, the buffer components should preferably not bereactive under reductive amination conditions. This exclude for instanceorganic buffer systems containing primary and—to lesser extend—secondaryamino groups. The skilled person will know which buffers are suitableand which are not. Some examples of suitable buffers are shown in table1 below:

TABLE 1 Buffers Common pKa at Buffer Name 25° C. Range Full CompoundName Bicine 8.35 7.6-9.0 N,N-bis(2-hydroxyethyl)glycine Hepes 7.486.8-8.2 4-2-hydroxyethyl-1- piperazineethanesulfonic acid TES 7.406.8-8.2 2-{[tris(hydroxymethyl)methyl]- amino}ethanesulfonic acid MOPS7.20 6.5-7.9 3-(N-morpholino)propanesulfonic acid PIPES 6.76 6.1-7.5piperazine-N,N′-bis(2-ethanesulfonic acid) MES 6.15 5.5-6.72-(N-morpholino)ethanesulfonic acid

By applying this method, GSC reagents modified with half-life extendingmoieties, having isomer free stable linkages can efficient be prepared,and isolated in a simple process that minimize the chance for hydrolysisof the CMP activation group.

By reacting either of said compounds with each other a HEP-GSC conjugatecomprising a 4-methylbenzoyl sublinker moiety may be created.

GSC may also be reacted with thiobutyrolactone, thereby creating a thiolmodified GSC molecule (GSC-SH). As demonstrated in the presentinvention, such reagents may be reacted with maleimide functionalizedHEP polymers to form HEP-GSC reagents. This synthesis route is depictedin FIG. 15. The resulting product has a linkage structure comprisingsuccinimide.

However, succinimide based (sub)linkages may undergo hydrolytic ringopening inter alia when the modified GSC reagent is stored in aqueoussolution for extended time periods and while the linkage may remainintact, the ring opening reaction will add undesirable heterogeneity inform of regio- and stereo-isomers.

Methods of Glycoconjugation

Conjugation of a HEP-GSC conjugate with a (poly)-peptide may be carriedout via a glycan present on residues in the (poly)-peptide backbone.This form of conjugation is also referred to as glycoconjugation.

Methods based on sialyltransferase have over the years proven to be mildand highly selective for modifying N-glycans or O-glycans on bloodcoagulation factors, such as coagulation factor FVII.

In contrast to conjugation methods based on cysteine alkylations, lysineacylations and similar conjugations involving amino acids in the proteinbackbone, conjugation via glycans is an appealing way of attachinglarger structures such as polymers of protein/peptide fragments tobioactive proteins with less disturbance of bioactivity. This is becauseglycans being highly hydrophilic generally tend to be oriented away fromthe protein surface and out in solution, leaving the binding surfacesthat are important for the proteins activity free.

The glycan may be naturally occurring or it may be inserted via e.g.insertion of an N-linked glycan using methods well known in the art.

GSC is a sialic acid derivative that can be transferred to glycoproteinsby the use of sialyltransferases. It can be selectively modified withsubstituents such as PEG on the glycyl amino group and still beenzymatically transferred to glycoproteins by use of sialyltransferases.GSC can be efficiently prepared by an enzymatic process in large scale(WO07056191).

Sialyltransferases

Sialyltransferases are a class of glycosyltransferases that transfersialic acid from naturally activated sialic acid (Sia)-CMP (cytidinemonophosphate) compounds to galactosyl-moieties on e.g. proteins. Manysialyltransferases (ST3GalIII, ST3GalI, ST6GalNAcI) are capable oftransfer of sialic acid-CMP (Sia-CMP) derivatives that have beenmodified on the C5 acetamido group inter alia with large groups such as40 kDa PEG (WO03031464). An extensive, but non-limited list of relevantsialyltransferases that can be used with the current invention isdisclosed in WO2006094810, which is hereby incorporated by reference inits entirety.

In one aspect of the present invention, terminal sialic acids onglycoproteins can be removed by sialidase treatment to provide asialoglycoproteins. Asialo glycoproteins and GSC modified with the half-lifeextending moiety together will act as substrates for sialyltransferases.The product of the reaction is a glycoprotein conjugate having thehalf-life extending moiety linked via an intact glycosyl linkinggroup—in this case an intact sialic acid linker group. A reaction schemewherein an asialo FVIIa glycoprotein is reacted with HEP-GSC in thepresence of sialyltransferase is shown in FIG. 18.

The term “sialic acid” refers to any member of a family of nine-carboncarboxylated sugars. The most common member of the sialic acid family isN-acetylneuraminic acid(2-keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactononulopyranos-1-onicacid (often abbreviated as Neu5Ac, NeuAc, NeuNAc, or NANA). A secondmember of the family is N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), inwhich the N-acetyl group of NeuNAc is hydroxylated. A third sialic acidfamily member is 2-keto-3-deoxy-nonulosonic acid (KDN) (Nadano et al.(1986) J. Biol. Chem. 261: 11550-11557; Kanamori et al., J. Biol. Chem.265: 21811-21819 (1990)). Also included are 9-substituted sialic acidssuch as a 9-O-C1-C6 acyl-Neu5Ac like 9-O-lactylNeu5Ac or9-O-acetyl-Neu5Ac. The synthesis and use of sialic acid compounds in asialylation procedure is disclosed in international applicationWO92/16640, published Oct. 1, 1992.

The term “sialic acid derivative” refers to sialic acids as definedabove that are modified with one or more chemical moieties. Themodifying group may for example be alkyl groups such as methyl groups,azido- and fluoro groups, or functional groups such as amino or thiolgroups that can function as handles for attaching other chemicalmoieties. Examples include 9-deoxy-9-fluoro-Neu5Ac and9-azido-9-deoxy-Neu5Ac. The term also encompasses sialic acids that lackone of more functional groups such as the carboxyl group or one or moreof the hydroxyl groups. Derivatives where the carboxyl group is replacedwith a carboxamide group or an ester group are also encompassed by theterm. The term also refers to sialic acids where one or more hydroxylgroups have been oxidized to carbonyl groups. Furthermore the termrefers to sialic acids that lack the C9 carbon atom or both the C9-C8carbon chain for example after oxidative treatment with periodate.

Glycyl sialic acid is a sialic acid derivative according to thedefinition above, where the N-acetyl group of NeuNAc is replaced with aglycyl group also known as an amino acetyl group. Glycyl sialic acid maybe represented with the following structure:

The term “CMP-activated” sialic acid or sialic acid derivatives refer toa sugar nucleotide containing a sialic acid moiety and a cytidinemonophosphate (CMP).

In the present description, the term “glycyl sialic acid cytidinemonophosphate” is used for describing GSC, and is a synonym foralternative naming of same CMP activated glycyl sialic acid. Alternativenaming include CMP-5′-glycyl sialic acid,cytidine-5′-monophospho-N-glycylneuraminic acid,cytidine-5′-monophospho-N-glycyl sialic acid.

The term “intact glycosyl linking group” refers to a linking group thatis derived from a glycosyl moiety in which the saccharide monomerinterposed between and covalently attached to the polypeptide and theHEP moiety is not degraded, e.g., oxidized, e.g., by sodiummetaperiodate during conjugate formation. “Intact glycosyl linkinggroups” may be derived from a naturally occurring oligosaccharide byaddition of glycosyl unites or removal of one or more glycosyl unit froma parent saccharide structure.

The term “asialo glycoprotein” is intended to include glycoproteinswherein one or more terminal sialic acid residues have been removed,e.g., by treatment with a sialidase or by chemical treatment, exposingat least one galactose or N-acetylgalactosamine residue from theunderlying “layer” of galactose or N-acetylgalactosamine (“exposedgalactose residue”).

Dotted lines in structure formulas denotes open valence bond (i.e. bondsthat connect the structures to other chemical moieties).

PEGylated Derivatives

“PEGylated Factor VII polypeptide variants/derivatives” according to thepresent invention may have one or more polyethylene glycol (PEG)molecules attached to any part of the FVII polypeptide including anyamino acid residue or carbohydrate moiety of the Factor VII polypeptide.Chemical and/or enzymatic methods can be employed for conjugating PEG orother half-life extending moieties to a glycan on the Factor VIIpolypeptide. An example of an enzymatic conjugation process is describede.g. in WO03031464. The glycan may be naturally occurring or it may beengineered as described above for HEP conjugates. “Cysteine-PEGylatedFactor VII polypeptide variants” according to the present invention haveone or more PEG molecules conjugated to a sulfhydryl group of a cysteineresidue present or introduced in the FVII polypeptide.

Fusion Proteins

Fusion proteins are proteins created through the in-frame joining of twoor more DNA sequences which originally encode separate proteins orpeptides or fragments thereof. Translation of the fusion protein DNAsequence will result in a single protein sequence which may havefunctional properties derived from each of the original proteins orpeptides. DNA sequences encoding fusion proteins may be createdartificially by standard molecular biology methods such as overlappingPCR or DNA ligation and the assembly is performed excluding the stopcodon in the first 5′-end DNA sequence while retaining the stop codon inthe 3′-end DNA sequence. The resulting fusion protein DNA sequence maybe inserted into an appropriate expression vector that supports theheterologous fusion protein expression in standard host organisms suchas bacteria, yeast, fungi, insect cells or mammalian cells.

Fusion proteins may contain a linker or spacer peptide sequence thatseparates the protein or peptide parts which define the fusion protein.

In one interesting embodiment of the invention, the Factor VIIpolypeptide is a fusion protein comprising a Factor VII polypeptide anda fusion partner protein/peptide, for example an Fc domain or analbumin.

Fc Fusion Protein

The term “Fc fusion protein” is herein meant to encompass Factor VIIpolypeptides of this invention fused to an Fc domain that can be derivedfrom any antibody isotype. An IgG Fc domain will often be preferred dueto the relatively long circulatory half-life of IgG antibodies. The Fcdomain may furthermore be modified in order to modulate certain effectorfunctions such as e.g. complement binding and/or binding to certain Fcreceptors. Fusion of FVII polypeptides with an Fc domain, which has thecapacity to bind to FcRn receptors, will generally result in a prolongedcirculatory half-life of the fusion protein compared to the half-life ofthe wt FVII polypeptides. Mutations in positions 234, 235 and 237 in anIgG Fc domain will generally result in reduced binding to the FcγRIreceptor and possibly also the FcγRIIa and the FcγRIII receptors. Thesemutations do not alter binding to the FcRn receptor, which promotes along circulatory half-life by an endocytic recycling pathway.Preferably, a modified IgG Fc domain of a fusion protein according tothe invention comprises one or more of the following mutations that willresult in decreased affinity to certain Fc receptors (L234A, L235E, andG237A) and in reduced C1q-mediated complement fixation (A330S andP331S), respectively. Alternatively, the Fc domain may be an IgG4 Fcdomain, preferably comprising the S241P/S228P mutation.

Production of Factor VII Polypeptides

Factor VII polypeptides, of the current invention, may be recombinantlyproduced using well known methods of production and purification; someexamples of these methods are described below; yet further examples ofmethods of production and purification are, inter alia, described inWO2007/031559.

In one aspect, the invention relates to a method for producing FactorVII polypeptides. The Factor VII polypeptides described herein may beproduced by means of recombinant nucleic acid techniques. In general, acloned human wild-type Factor VII nucleic acid sequence is modified toencode the desired protein. This modified sequence is then inserted intoan expression vector, which is in turn transformed or transfected intohost cells. Higher eukaryotic cells, in particular cultured mammaliancells, are preferred as host cells.

In a further aspect, the invention relates to a transgenic animalcontaining and expressing the polynucleotide construct.

The complete nucleotide and amino acid sequences for human wild-typeFactor VII are known (see U.S. Pat. No. 4,784,950, where the cloning andexpression of recombinant human Factor VII is described).

The amino acid sequence alterations may be accomplished by a variety ofknow techniques. Modification of the nucleic acid sequence may be bysite-specific mutagenesis. Techniques for site-specific mutagenesis arewell known in the art and are described in, for example, Zoller andSmith (DNA 3:479-488, 1984) or “Splicing by extension overlap”, Hortonet al., Gene 77, 1989, pp. 61-68. Thus, using the nucleotide and aminoacid sequences of Factor VII, one may introduce the alteration(s) ofchoice. Likewise, procedures for preparing a DNA construct usingpolymerase chain reaction using specific primers are well known topersons skilled in the art (cf. PCR Protocols, 1990, Academic Press, SanDiego, Calif., USA).

The nucleic acid construct encoding the Factor VII polypeptide of theinvention may suitably be of genomic or cDNA origin, The nucleic acidconstruct encoding the Factor VII polypeptide may also be preparedsynthetically by established standard methods, e.g. the phosphoamiditemethod described by Beaucage and Caruthers, Tetrahedron Letters 22(1981), 1859-1869, The DNA sequences encoding the human Factor VIIpolypeptides may also be prepared by polymerase chain reaction usingspecific primers, for instance as described in U.S. Pat. No. 4,683,202,Saiki et al., Science 239 (1988), 487-491, or Sambrook et al., supra.

Furthermore, the nucleic acid construct may be of mixed synthetic andgenomic, mixed synthetic and cDNA or mixed genomic and cDNA originprepared by ligating fragments of synthetic, genomic or cDNA origin (asappropriate), the fragments corresponding to various parts of the entirenucleic acid construct, in accordance with standard techniques.

The nucleic acid construct is preferably a DNA construct. DNA sequencesfor use in producing Factor VII polypeptides according to the presentinvention will typically encode a pre-pro polypeptide at theamino-terminus of Factor VII to obtain proper posttranslationalprocessing (e.g. gamma-carboxylation of glutamic acid residues) andsecretion from the host cell. The pre-pro polypeptide may be that ofFactor VII or another vitamin K-dependent plasma protein, such as FactorIX, Factor X, prothrombin, protein C or protein S. As will beappreciated by those skilled in the art, additional modifications can bemade in the amino acid sequence of the Factor VII polypeptides wherethose modifications do not significantly impair the ability of theprotein to act as a coagulant.

The DNA sequences encoding the human Factor VII polypeptides are usuallyinserted into a recombinant vector which may be any vector, which mayconveniently be subjected to recombinant DNA procedures, and the choiceof vector will often depend on the host cell into which it is to beintroduced. Thus, the vector may be an autonomously replicating vector,i.e. a vector, which exists as an extrachromosomal entity, thereplication of which is independent of chromosomal replication, e.g. aplasmid. Alternatively, the vector may be one which, when introducedinto a host cell, is integrated into the host cell genome and replicatedtogether with the chromosome(s) into which it has been integrated.

The vector is preferably an expression vector in which the DNA sequenceencoding the human Factor VII polypeptides is operably linked toadditional segments required for transcription of the DNA. In general,the expression vector is derived from plasmid or viral DNA, or maycontain elements of both. The term, “operably linked” indicates that thesegments are arranged so that they function in concert for theirintended purposes, e.g. transcription initiates in a promoter andproceeds through the DNA sequence coding for the polypeptide.

Expression vectors for use in expressing Factor VIIa polypeptidevariants will comprise a promoter capable of directing the transcriptionof a cloned gene or cDNA. The promoter may be any DNA sequence, whichshows transcriptional activity in the host cell of choice and may bederived from genes encoding proteins either homologous or heterologousto the host cell.

Examples of suitable promoters for directing the transcription of theDNA encoding the human Factor VII polypeptide in mammalian cells are theSV40 promoter (Subramani et al., Mol. Cell Biol. 1 (1981), 854-864), theMT-1 (metallothionein gene) promoter (Palmiter et al., Science 222(1983), 809-814), the CMV promoter (Boshart et al., Cell 41:521-530,1985) or the adenovirus 2 major late promoter (Kaufman and Sharp, Mol.Cell. Biol, 2:1304-1319, 1982).

The DNA sequences encoding the Factor VII polypeptides may also, ifnecessary, be operably connected to a suitable terminator, such as thehuman growth hormone terminator (Palmiter et al., Science 222, 1983, pp.809-814) or the TPI1 (Alber and Kawasaki, J. Mol. Appl. Gen. 1, 1982,pp. 419-434) or ADH3 (McKnight et al., The EMBO J. 4, 1985, pp.2093-2099) terminators. Expression vectors may also contain a set of RNAsplice sites located downstream from the promoter and upstream from theinsertion site for the Factor VII sequence itself. Preferred RNA splicesites may be obtained from adenovirus and/or immunoglobulin genes. Alsocontained in the expression vectors is a polyadenylation signal locateddownstream of the insertion site. Particularly preferred polyadenylationsignals include the early or late polyadenylation signal from SV40(Kaufman and Sharp, ibid.), the polyadenylation signal from theadenovirus 5 EIb region, the human growth hormone gene terminator(DeNoto et al. Nucl. Acids Res. 9:3719-3730, 1981) or thepolyadenylation signal from the human Factor VII gene or the bovineFactor VII gene. The expression vectors may also include a noncodingviral leader sequence, such as the adenovirus 2 tripartite leader,located between the promoter and the RNA splice sites; and enhancersequences, such as the SV40 enhancer.

To direct the Factor VII polypeptides of the present invention into thesecretory pathway of the host cells, a secretory signal sequence (alsoknown as a leader sequence, prepro sequence or pre sequence) may beprovided in the recombinant vector. The secretory signal sequence isjoined to the DNA sequences encoding the human Factor VII polypeptidesin the correct reading frame. Secretory signal sequences are commonlypositioned 5′ to the DNA sequence encoding the peptide. The secretorysignal sequence may be that, normally associated with the protein or maybe from a gene encoding another secreted protein.

The procedures used to ligate the DNA sequences coding for the FactorVII polypeptides, the promoter and optionally the terminator and/orsecretory signal sequence, respectively, and to insert them intosuitable vectors containing the information necessary for replication,are well known to persons skilled in the art (cf., for instance,Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor, N.Y., 1989).

Methods of transfecting mammalian cells and expressing DNA sequencesintroduced in the cells are described in e.g. Kaufman and Sharp, J. Mol.Biol. 159 (1982), 601-621; Southern and Berg, J. Mol. Appl. Genet. 1(1982), 327-341; Loyter et al., Proc. Natl. Acad. Sci. USA 79 (1982),422-426; Wigler et al., Cell 14 (1978), 725; Corsaro and Pearson,Somatic Cell Genetics 7 (1981), 603, Graham and van der Eb, Virology 52(1973), 456; and Neumann et al., EMBO J. 1 (1982), 841-845.

Cloned DNA sequences are introduced into cultured mammalian cells by,for example, calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725-732, 1978; Corsaro and Pearson, Somatic Cell Genetics7:603-616, 1981; Graham and Van der Eb, Virology 52d:456-467, 1973) orelectroporation (Neumann et al., EMBO J. 1:841-845, 1982). To identifyand select cells that express the exogenous DNA, a gene that confers aselectable phenotype (a selectable marker) is generally introduced intocells along with the gene or cDNA of interest. Preferred selectablemarkers include genes that confer resistance to drugs such as neomycin,hygromycin, and methotrexate. The selectable marker may be anamplifiable selectable marker. A preferred amplifiable selectable markeris a dihydrofolate reductase (DHFR) sequence. Selectable markers may beintroduced into the cell on a separate plasmid at the same time as thegene of interest, or they may be introduced on the same plasmid. If, onthe same plasmid, the selectable marker and the gene of interest may beunder the control of different promoters or the same promoter, thelatter arrangement producing a dicistronic message. Constructs of thistype are known in the art (for example, Levinson and Simonsen, U.S. Pat.No. 4,713,339). It may also be advantageous to add additional DNA, knownas “carrier DNA,” to the mixture that is introduced into the cells.

After the cells have taken up the DNA, they are grown in an appropriategrowth medium, typically 1-2 days, to begin expressing the gene ofinterest. As used herein the term “appropriate growth medium” means amedium containing nutrients and other components required for the growthof cells and the expression of the Factor VII polypeptides of interest.Media generally include a carbon source, a nitrogen source, essentialamino acids, essential sugars, vitamins, salts, phospholipids, proteinand growth factors. For production of gamma-carboxylated proteins, themedium will contain vitamin K, preferably at a concentration of about0.1 μg/ml to about 5 μg/ml. Drug selection is then applied to select forthe growth of cells that are expressing the selectable marker in astable fashion. For cells that have been transfected with an amplifiableselectable marker the drug concentration may be increased to select foran increased copy number of the cloned sequences, thereby increasingexpression levels. Clones of stably transfected cells are then screenedfor expression of the human Factor VII polypeptide of interest.

The host cell into which the DNA sequences encoding the Factor VIIpolypeptides is introduced may be any cell, which is capable ofproducing the posttranslational modified human Factor VII polypeptidesand includes yeast, fungi and higher eucaryotic cells.

Examples of mammalian cell lines for use in the present invention arethe Chinese Hamster Ovary (CHO) cells (e.g. ATCC CCL 61), CHO DUKX cells(Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220, 1980).),baby hamster kidney (BHK) and 293 (ATCC CRL 1573; Graham et al., J. Gen.Virol. 36:59-72, 1977) cell lines.

The transformed or transfected host cell described above is thencultured in a suitable nutrient medium under conditions permittingexpression of the Factor VII polypeptide after which all or part of theresulting peptide may be recovered from the culture. The medium used toculture the cells may be any conventional medium suitable for growingthe host cells, such as minimal or complex media containing appropriatesupplements. Suitable media are available from commercial suppliers ormay be prepared according to published recipes (e.g. in catalogues ofthe American Type Culture Collection). The Factor VII polypeptideproduced by the cells may then be recovered from the culture medium byconventional procedures including separating the host cells from themedium by centrifugation or filtration, precipitating the proteinaqueouscomponents of the supernatant or filtrate by means of a salt, e.g.ammonium sulphate, purification by a variety of chromatographicprocedures, e.g. ion exchange chromatography, gelfiltrationchromatography, affinity chromatography, or the like, dependent on thetype of polypeptide in question.

Transgenic animal technology may be employed to produce the Factor VIIpolypeptides of the invention. It is preferred to produce the proteinswithin the mammary glands of a host female mammal. Expression in themammary gland and subsequent secretion of the protein of interest intothe milk overcomes many difficulties encountered in isolating proteinsfrom other sources. Milk is readily collected, available in largequantities, and biochemically well characterized. Furthermore, the majormilk proteins are present in milk at high concentrations (typically fromabout 1 to 15 g/l).

From a commercial point of view, it is clearly preferable to use as thehost a species that has a large milk yield. While smaller animals suchas mice and rats can be used (and are preferred at the proof ofprinciple stage), it is preferred to use livestock mammals including,but not limited to, pigs, goats, sheep and cattle. Sheep areparticularly preferred due to such factors as the previous history oftransgenesis in this species, milk yield, cost and the readyavailability of equipment for collecting sheep milk (see, for example,WO 88/00239 for a comparison of factors influencing the choice of hostspecies). It is generally desirable to select a breed of host animalthat has been bred for dairy use, such as East Friesland sheep, or tointroduce dairy stock by breeding of the transgenic line at a laterdate. In any event, animals of known, good health status should be used.

To obtain expression in the mammary gland, a transcription promoter froma milk protein gene is used. Milk protein genes include those genesencoding caseins (see U.S. Pat. No. 5,304,489), beta-lactoglobulin,a-lactalbumin, and whey acidic protein. The beta-lactoglobulin (BLG)promoter is preferred. In the case of the ovine beta-lactoglobulin gene,a region of at least the proximal 406 bp of 5′ flanking sequence of thegene will generally be used, although larger portions of the 5′ flankingsequence, up to about 5 kbp, are preferred, such as a ˜4.25 kbp DNAsegment encompassing the 5′ flanking promoter and non-coding portion ofthe beta-lactoglobulin gene (see Whitelaw et al., Biochem. J. 286: 31-39(1992)). Similar fragments of promoter DNA from other species are alsosuitable.

Other regions of the beta-lactoglobulin gene may also be incorporated inconstructs, as may genomic regions of the gene to be expressed. It isgenerally accepted in the art that constructs lacking introns, forexample, express poorly in comparison with those that contain such DNAsequences (see Brinster et al., Proc. Natl. Acad. Sci. USA 85: 836-840(1988); Palmiter et al., Proc. Natl. Acad. Sci. USA 88: 478-482 (1991);Whitelaw et al., Transgenic Res. 1: 3-13 (1991); WO 89/01343; and WO91/02318, each of which is incorporated herein by reference). In thisregard, it is generally preferred, where possible, to use genomicsequences containing all or some of the native introns of a geneencoding the protein or polypeptide of interest, thus the furtherinclusion of at least some introns from, e.g, the beta-lactoglobulingene, is preferred. One such region is a DNA segment that provides forintron splicing and RNA polyadenylation from the 3′ non-coding region ofthe ovine beta-lactoglobulin gene. When substituted for the natural 3′non-coding sequences of a gene, this ovine beta-lactoglobulin segmentcan both enhance and stabilize expression levels of the protein orpolypeptide of interest. Within other embodiments, the regionsurrounding the initiation ATG of the variant Factor VII sequence isreplaced with corresponding sequences from a milk specific protein gene.Such replacement provides a putative tissue-specific initiationenvironment to enhance expression. It is convenient to replace theentire variant Factor VII pre-pro and 5′ non-coding sequences with thoseof, for example, the BLG gene, although smaller regions may be replaced.

For expression of Factor VII polypeptides in transgenic animals, a DNAsegment encoding variant Factor VII is operably linked to additional DNAsegments required for its expression to produce expression units. Suchadditional segments include the above-mentioned promoter, as well assequences that provide for termination of transcription andpolyadenylation of mRNA. The expression units will further include a DNAsegment encoding a secretory signal sequence operably linked to thesegment encoding modified Factor VII. The secretory signal sequence maybe a native Factor VII secretory signal sequence or may be that ofanother protein, such as a milk protein (see, for example, von Heijne,Nucl. Acids Res. 14: 4683-4690 (1986); and Meade et al., U.S. Pat. No.4,873,316, which are incorporated herein by reference).

Construction of expression units for use in transgenic animals isconveniently carried out by inserting a variant Factor VII sequence intoa plasmid or phage vector containing the additional DNA segments,although the expression unit may be constructed by essentially anysequence of ligations. It is particularly convenient to provide a vectorcontaining a DNA segment encoding a milk protein and to replace thecoding sequence for the milk protein with that of a Factor VII variant;thereby creating a gene fusion that includes the expression controlsequences of the milk protein gene. In any event, cloning of theexpression units in plasmids or other vectors facilitates theamplification of the variant Factor VII sequence. Amplification isconveniently carried out in bacterial (e.g. E. coli) host cells, thusthe vectors will typically include an origin of replication and aselectable marker functional in bacterial host cells. The expressionunit is then introduced into fertilized eggs (including early-stageembryos) of the chosen host species. Introduction of heterologous DNAcan be accomplished by one of several routes, including microinjection(e.g. U.S. Pat. No. 4,873,191), retroviral infection (Jaenisch, Science240: 1468-1474 (1988)) or site-directed integration using embryonic stem(ES) cells (reviewed by Bradley et al., Bio/Technology 10: 534-539(1992)). The eggs are then implanted into the oviducts or uteri ofpseudopregnant females and allowed to develop to term. Offspringcarrying the introduced DNA in their germ line can pass the DNA on totheir progeny in the normal, Mendelian fashion, allowing the developmentof transgenic herds. General procedures for producing transgenic animalsare known in the art (see, for example, Hogan et al., Manipulating theMouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory, 1986;Simons et al., Bio/Technology 6: 179-183 (1988); Wall et al., Biol.Reprod. 32: 645-651 (1985); Buhler et al., Bio/Technology 8: 140-143(1990); Ebert et al., Bio/Technology 9: 835-838 (1991); Krimpenfort etal., Bio/Technology 9: 844-847 (1991); Wall et al., J. Cell. Biochem.49: 113-120 (1992); U.S. Pat. No. 4,873,191; U.S. Pat. No. 4,873,316; WO88/00239, WO 90/05188, WO 92/11757; and GB 87/00458). Techniques forintroducing foreign DNA sequences into mammals and their germ cells wereoriginally developed in the mouse (see, e.g., Gordon et al., Proc. Natl.Acad. Sci. USA 77: 7380-7384 (1980); Gordon and Ruddle, Science 214:1244-1246 (1981); Palmiter and Brinster, Cell 41: 343-345 (1985);Brinster et al., Proc. Natl. Acad. Sci. USA 82: 4438-4442 (1985); andHogan et al. (ibid.)). These techniques were subsequently adapted foruse with larger animals, including livestock species (see, e.g., WO88/00239, WO 90/05188, and WO 92/11757; and Simons et al.,Bio/Technology 6: 179-183 (1988)). To summarise, in the most efficientroute used to date in the generation of transgenic mice or livestock,several hundred linear molecules of the DNA of interest are injectedinto one of the pro-nuclei of a fertilized egg according to establishedtechniques. Injection of DNA into the cytoplasm of a zygote can also beemployed.

Purification

The Factor VII polypeptides of the invention are recovered from cellculture medium. The Factor VII polypeptides of the present invention maybe purified by a variety of procedures known in the art including, butnot limited to, chromatography (e.g., ion exchange, affinity,hydrophobic, chromatofocusing, and size exclusion), electrophoreticprocedures (e.g., preparative isoelectric focusing (IEF), differentialsolubility (e.g., ammonium sulfate precipitation), or extraction (see,e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989). Preferably, Factor VII polypeptides may bepurified by affinity chromatography on an anti-Factor VII antibodycolumn. The use of calcium-dependent monoclonal antibodies, as describedby Wakabayashi et al., J. Biol. Chem. 261:11097-11108, (1986) and Thimet al., Biochemistry 27: 7785-7793, (1988), is particularly preferred.Additional purification may be achieved by conventional chemicalpurification means, such as high performance liquid chromatography.Other methods of purification, including barium citrate precipitation,are known in the art, and may be applied to the purification of thenovel Factor VII polypeptides described herein (see, for example,Scopes, R., Protein Purification, Springer-Verlag, N.Y., 1982).

For therapeutic purposes it is preferred that the Factor VIIpolypeptides of the invention are substantially pure. Thus, in apreferred embodiment of the invention the Factor VII polypeptides of theinvention are purified to at least about 90 to 95% homogeneity,preferably to at least about 98% homogeneity. Purity may be assessed byseveral methods known in the art e.g. HPLC, gel electrophoresis andamino-terminal amino acid sequencing.

The Factor VII polypeptide is cleaved at its activation site in order toconvert it to its two-chain form. Activation may be carried outaccording to procedures known in the art, such as those disclosed byOsterud, et al., Biochemistry 11:2853-2857 (1972); Thomas, U.S. Pat. No.4,456,591; Hedner and Kisiel, J. Clin. Invest. 71:1836-1841 (1983); orKisiel and Fujikawa, Behring Inst. Mitt. 73:29-42 (1983). Alternatively,as described by Bjoern et al. (Research Disclosure, 269 September 1986,pp. 564-565), Factor VII may be activated by passing it through anion-exchange chromatography column, such as Mono Q® (Pharmacia fineChemicals) or the like. The resulting activated Factor VII variant maythen be formulated and administered as described below.

Assays

Provided herein are suitable in vitro proteolytic and antithrombinreactivity assays for selecting preferred Factor VII polypeptidesaccording to the invention. Such assays are described in detail inExample 5. Briefly, the assays can be performed as simple preliminary invitro tests, as follows:

The proteolytic activity of FVIIa polypeptides can be measured using thephysiological substrate plasma-derived factor X (X) as substrate atphysiological pH and in the presence of calcium and vesicles composed ofphosphatidyl choline (PC) and phosphatidyl serine (PS) to support thereaction. The assay is performed by incubating FVIIa with FX at asubstrate concentration below Km for the reaction and for a periodsufficient long to allow for the generation of measurable amounts of FXawhile keeping the conversion of FX below 20%. The generated FXa isquantified after the addition of a suitable chromogenic substrate suchas S-2765 and reported relative to that of wild-type FVIIa followingnormalisation according to the concentration of the FVIIa varianttested.

The antithrombin reactivity of the FVIIa polypeptides can be measured atphysiological pH under pseudo-first order conditions in the presence ofexcess plasma-derived antithrombin, low molecular weight (LMW) heparinand calcium. Residual FVIIa activity is measured discontinuouslythroughout the time course of the inhibition reaction using achromogenic substrate such as S-2288. The rate of inhibition is obtainedby non-linear least-squares fitting of data to a single exponentialdecay function and reported relative to that of wild-type FVIIafollowing normalisation of inhibition rates according to theantithrombin concentration used. The kinetic characterisation ofheparin-catalyzed and uncatalyzed inhibition of blood coagulationproteinases by antithrombinis is described in Olson et al. (1993),Methods Enzymol. 222, 525-559.

Pharmaceutical Compositions

In one aspect, the present invention relates to compositions andformulations comprising a Factor VII polypeptide of the invention. Forexample, the invention provides a pharmaceutical composition thatcomprises a Factor VII polypeptide of the invention, formulated togetherwith a pharmaceutically acceptable carrier.

Accordingly, one object of the invention is to provide a pharmaceuticalformulation comprising a Factor VII polypeptide which is present in aconcentration from 0.25 mg/ml to 100 mg/ml, and wherein said formulationhas a pH from 2.0 to 10.0. The formulation may further comprise one ormore of a buffer system, a preservative, a tonicity agent, a chelatingagent, a stabilizer, an antioxidant or a surfactant, as well as variouscombinations thereof. The use of preservatives, isotonic agents,chelating agents, stabilizers, antioxidant and surfactants inpharmaceutical compositions is well-known to the skilled person.Reference may be made to Remington: The Science and Practice ofPharmacy, 19th edition, 1995.

In one embodiment, the pharmaceutical formulation is an aqueousformulation. Such a formulation is typically a solution or a suspension,but may also include colloids, dispersions, emulsions, and multi-phasematerials. The term “aqueous formulation” is defined as a formulationcomprising at least 50% w/w water. Likewise, the term “aqueous solution”is defined as a solution comprising at least 50% w/w water, and the term“aqueous suspension” is defined as a suspension comprising at least 50%w/w water.

In another embodiment, the pharmaceutical formulation is a freeze-driedformulation, to which the physician or the patient adds solvents and/ordiluents prior to use.

In a further aspect, the pharmaceutical formulation comprises an aqueoussolution of a Factor VII polypeptide, and a buffer, wherein thepolypeptide is present in a concentration from 1 mg/ml or above, andwherein said formulation has a pH from about 2.0 to about 10.0.

In a further aspect, the pharmaceutical formulation may be any one ofthose disclosed in WO2014/057069, which is herein incorporated byreference; or it may be the formulation described in Example 18.

A Factor VII polypeptide of the invention may be administeredparenterally, such as intravenously, such as intramuscularly, such assubcutaneously. Alternatively, a FVII polypeptide of the invention maybe administered via a non-parenteral route, such as perorally ortopically. An polypeptide of the invention may be administeredprophylactically. An polypeptide of the invention may be administeredtherapeutically (on demand).

Therapeutic Uses

In a broad aspect, a Factor VII polypeptide of the present invention ora pharmaceutical formulation comprising said polypeptide, may be used asa medicament.

In one aspect, a Factor VII polypeptide of the present invention or apharmaceutical formulation comprising said polypeptide, may be used totreat a subject with a coagulopathy.

In another aspect, a Factor VII polypeptide of the present invention ora pharmaceutical formulation comprising said polypeptide may be used forthe preparation of a medicament for the treatment of bleeding disordersor bleeding episodes or for the enhancement of the normal haemostaticsystem.

In a further aspect, a Factor VII polypeptide of the present inventionor a pharmaceutical formulation comprising said polypeptide may be usedfor the treatment of haemophilia A, haemophilia B or haemophilia A or Bwith acquired inhibitors.

In another aspect, a Factor VII polypeptide of the present invention ora pharmaceutical formulation comprising said polypeptide may be used ina method for the treatment of bleeding disorders or bleeding episodes ina subject or for the enhancement of the normal haemostatic system, themethod comprising administering a therapeutically or prophylacticallyeffective amount of a Factor VII polypeptide of the present invention toa subject in need thereof.

The term “subject”, as used herein, includes any human patient, ornon-human vertebrates.

The term “treatment”, as used herein, refers to the medical therapy ofany human or other vertebrate subject in need thereof. Said subject isexpected to have undergone physical examination by a medicalpractitioner, or a veterinary medical practitioner, who has given atentative or definitive diagnosis which would indicate that the use ofsaid specific treatment is beneficial to the health of said human orother vertebrate. The timing and purpose of said treatment may vary fromone individual to another, according to the status quo of the subject'shealth. Thus, said treatment may be prophylactic, palliative,symptomatic and/or curative. In terms of the present invention,prophylactic, palliative, symptomatic and/or curative treatments mayrepresent separate aspects of the invention.

The term “coagulopathy”, as used herein, refers to an increasedhaemorrhagic tendency which may be caused by any qualitative orquantitative deficiency of any pro-coagulative component of the normalcoagulation cascade, or any upregulation of fibrinolysis. Suchcoagulopathies may be congenital and/or acquired and/or iatrogenic andare identified by a person skilled in the art. Non-limiting examples ofcongenital hypocoagulopathies are haemophilia A, haemophilia B, FactorVII deficiency, Factor X deficiency, Factor XI deficiency, vonWillebrand's disease and thrombocytopenias such as Glanzmann'sthombasthenia and Bernard-Soulier syndrome. The clinical severity ofhaemophilia A or B is determined by the concentration of functionalunits of FIX/Factor VIII in the blood and is classified as mild,moderate, or severe. Severe haemophilia is defined by a clotting factorlevel of <0.01 U/ml corresponding to <1% of the normal level, whilepeople with moderate and mild haemophilia have levels from 1-5% and >5%,respectively. Haemophilia A with “inhibitors” (that is, allo-antibodiesagainst factor VIII) and haemophilia B with “inhibitors” (that is,allo-antibodies against factor IX) are non-limiting examples ofcoagulopathies that are partly congenital and partly acquired.

A non-limiting example of an acquired coagulopathy is serine proteasedeficiency caused by vitamin K deficiency; such vitamin K-deficiency maybe caused by administration of a vitamin K antagonist, such as warfarin.Acquired coagulopathy may also occur following extensive trauma. In thiscase otherwise known as the “bloody vicious cycle”, it is characterisedby haemodilution (dilutional thrombocytopaenia and dilution of clottingfactors), hypothermia, consumption of clotting factors and metabolicderangements (acidosis). Fluid therapy and increased fibrinolysis mayexacerbate this situation. Said haemorrhage may be from any part of thebody.

A non-limiting example of an iatrogenic coagulopathy is an overdosage ofanticoagulant medication—such as heparin, aspirin, warfarin and otherplatelet aggregation inhibitors—that may be prescribed to treatthromboembolic disease. A second, non-limiting example of iatrogeniccoagulopathy is that which is induced by excessive and/or inappropriatefluid therapy, such as that which may be induced by a blood transfusion.

In one embodiment of the current invention, haemorrhage is associatedwith haemophilia A or B. In another embodiment, haemorrhage isassociated with haemophilia A or B with acquired inhibitors. In anotherembodiment, haemorrhage is associated with thrombocytopenia. In anotherembodiment, haemorrhage is associated with von Willebrand's disease. Inanother embodiment, haemorrhage is associated with severe tissue damage.In another embodiment, haemorrhage is associated with severe trauma. Inanother embodiment, haemorrhage is associated with surgery. In anotherembodiment, haemorrhage is associated with haemorrhagic gastritis and/orenteritis. In another embodiment, the haemorrhage is profuse uterinebleeding, such as in placental abruption. In another embodiment,haemorrhage occurs in organs with a limited possibility for mechanicalhaemostasis, such as intracranially, intraaurally or intraocularly. Inanother embodiment, haemorrhage is associated with anticoagulanttherapy.

The invention is further described by the following non-limiting list ofembodiments:

Embodiment 1

Factor VII polypeptide comprising two or more substitutions relative tothe amino acid sequence of human Factor VII (SEQ ID NO: 1), wherein T293is replaced by Lys (K), Arg (R), Tyr (Y) or Phe (F) and L288 is replacedby Phe (F), Tyr (Y), Asn (N), Ala (A) or Trp (W) and/or W201 is replacedby Arg (R), Met (M), or Lys (K) and/or K337 is replaced by Ala (A) orGly (G); optionally, where Q176 is replaced by Lys (K), Arg (R) or Asn(N); or Q286 is replaced by Asn (N).

Embodiment 1(i)

Factor VII polypeptide according to embodiment 1, wherein T293 isreplaced by Lys (K), Arg (R), Tyr (Y) or Phe (F); and L288 is replacedby Phe (F), Tyr (Y), Asn (N), Ala (A) or Trp (W) and/or W201 is replacedby Arg (R), Met (M) or Lys (K) and/or K337 is replaced by Ala (A) or Gly(G).

Embodiment 1(ii)

Factor VII polypeptide according to embodiment 1, wherein L288 isreplaced by Phe (F), Tyr (Y), Asn (N) or Ala (A).

Embodiment 1(iii)

Factor VII polypeptide according to embodiment 1, wherein W201 isreplaced by Arg (R), Met (M) or Lys (K).

Embodiment 1(iv)

Factor VII polypeptide according to embodiment 1, wherein K337 isreplaced by Ala (A) or Gly (G).

Embodiment 2

Factor VII polypeptide according to embodiment 1, wherein T293 isreplaced by Lys (K), Arg (R), Tyr (Y) or Phe (F).

Embodiment 2(i)

Factor VII polypeptide according to any one of embodiments 1-2, whereinT293 is replaced by Lys (K), Arg (R), Tyr (Y) or Phe (F) and L288 isreplaced by Phe (F), Tyr (Y), Asn (N), Ala (A) or Trp (W).

Embodiment 2(ii)

Factor VII polypeptide according to any one of embodiments 1-2, whereinT293 is replaced by Lys (K) and L288 is replaced by Phe (F).

Embodiment 2(iii)

Factor VII polypeptide according to any one of embodiments 1-2, whereinT293 is replaced by Lys (K) and L288 is replaced by Tyr (Y).

Embodiment 2(iv)

Factor VII polypeptide according to any one of embodiments 1-2, whereinT293 is replaced by Lys (K) and L288 is replaced by Asn (N).

Embodiment 2(v)

Factor VII polypeptide according to any one of embodiments 1-2, whereinT293 is replaced by Lys (K) and L288 is replaced by Ala (A).

Embodiment 2(vi)

Factor VII polypeptide according to any one of embodiments 1-2, whereinT293 is replaced by Lys (K) and L288 is replaced by Trp (W).

Embodiment 2(vii)

Factor VII polypeptide according to any one of embodiments 1-2, whereinT293 is replaced by Arg (R) and L288 is replaced by Phe (F).

Embodiment 2(viii)

Factor VII polypeptide according to any one of embodiments 1-2, whereinT293 is replaced by Arg (R) and L288 is replaced by Tyr (Y).

Embodiment 2(ix)

Factor VII polypeptide according to any one of embodiments 1-2, whereinT293 is replaced by Arg (R) and L288 is replaced by Asn (N).

Embodiment 2(x)

Factor VII polypeptide according to any one of embodiments 1-2, whereinT293 is replaced by Arg (R) and L288 is replaced by Ala (A).

Embodiment 2(xi)

Factor VII polypeptide according to any one of embodiments 1-2, whereinT293 is replaced by Arg (R) and L288 is replaced by Trp (W).

Embodiment 2(xii)

Factor VII polypeptide according to any one of embodiments 1-2, whereinT293 is replaced by Tyr (Y); and L288 is replaced by Phe (F).

Embodiment 2(xiii)

Factor VII polypeptide according to any one of embodiments 1-2, whereinT293 is replaced by Tyr (Y); and L288 is replaced by Tyr (Y).

Embodiment 2(xiv)

Factor VII polypeptide according to any one of embodiments 1-2, whereinT293 is replaced by Tyr (Y) and L288 is replaced by Asn (N).

Embodiment 2(xv)

Factor VII polypeptide according to any one of embodiments 1-2, whereinT293 is replaced by Tyr (Y) and L288 is replaced by Ala (A).

Embodiment 2(xvi)

Factor VII polypeptide according to any one of embodiments 1-2, whereinT293 is replaced by Tyr (Y) and L288 is replaced by Trp (W).

Embodiment 2(xvii)

Factor VII polypeptide according to any one of embodiments 1-2, whereinT293 is replaced by Phe (F) and L288 is replaced by Phe (F).

Embodiment 2(xviii)

Factor VII polypeptide according to any one of embodiments 1-2, whereinT293 is replaced by Phe (F) and L288 is replaced by Tyr (Y).

Embodiment 2(xix)

Factor VII polypeptide according to any one of embodiments 1-2, whereinT293 is replaced by Phe (F) and L288 is replaced by Asn (N).

Embodiment 2(xx)

Factor VII polypeptide according to any one of embodiments 1-2, whereinT293 is replaced by Phe (F) and L288 is replaced by Ala (A).

Embodiment 2(xxi)

Factor VII polypeptide according to any one of embodiments 1-2, whereinT293 is replaced by Phe (F) and L288 is replaced by Trp (W).

Embodiment 2(xxii)

Factor VII polypeptide according to any one of embodiments 1-2, whereinT293 is replaced by Lys (K) and K337 is replaced by Ala (A).

Embodiment 2(xxiii)

Factor VII polypeptide according to any one of embodiments 1-2, whereinT293 is replaced by Arg (R) and K337 is replaced by Ala (A).

Embodiment 2(xxiv)

Factor VII polypeptide according to any one of embodiments 1-2, whereinT293 is replaced by Tyr (Y) and K337 is replaced by Ala (A).

Embodiment 2(xxv)

Factor VII polypeptide according to any one of embodiments 1-2, whereinT293 is replaced by Phe (F) and K337 is replaced by Ala (A).

Embodiment 2(xxvi)

Factor VII polypeptide according to any one of embodiments 1-2, whereinT293 is replaced by Lys (K) and K337 is replaced by Gly (G).

Embodiment 2(xxvii)

Factor VII polypeptide according to any one of embodiments 1-2, whereinT293 is replaced by Arg (R) and K337 is replaced by Gly (G).

Embodiment 2(xxviii)

Factor VII polypeptide according to any one of embodiments 1-2, whereinT293 is replaced by Tyr (Y) and K337 is replaced by Gly (G).

Embodiment 2(xxix)

Factor VII polypeptide according to any one of embodiments 1-2, whereinT293 is replaced by Phe (F) and K337 is replaced by Gly (G).

Embodiment 2(xxx)

Factor VII polypeptide according to any one of embodiments 2(ii)-2(xxii)wherein K337 is replaced by Ala (A).

Embodiment 3

Factor VII polypeptide according to embodiment 2, wherein thepolypeptide comprises one of the following groups of substitutions:L288F/T293K, L288F/T293K/K337A, L288F/T293K/L305V, L288F/T293K/L305I,L288F/T293R, L288F/T293R/K337A, L288F/T293R/L305V, L288F/T293R/L305I,L288F/T293Y, L288F/T293Y/K337A, L288F/T293Y/L305V, L288F/T293Y/L305I,L288F/T293F, L288F/T293F/K337A, L288F/T293F/L305V, L288F/T293F/L305I,L288Y/T293K, L288Y/T293K/K337A, L288Y/T293K/L305V, L288Y/T293K/L305I,L288Y/T293R, L288Y/T293R/K337A, L288Y/T293R/L305V, L288Y/T293R/L305I,L288Y/T293Y, L288Y/T293Y/K337A, L288Y/T293Y/L305V, L288Y/T293Y/L305I,L288Y/T293F, L288Y/T293F/K337A, L288Y/T293F/L305V, L288Y/T293F/L305I,L288N/T293K, L288N/T293K/K337A, L288N/T293K/L305V, L288N/T293K/L305I,L288N/T293R, L288N/T293R/K337A, L288N/T293R/L305V, L288N/T293R/L305I,L288N/T293Y, L288N/T293Y/K337A, L288N/T293Y/L305V, L288N/T293Y/L305I,L288N/T293F, L288N/T293F/K337A, L288N/T293F/L305V, L288N/T293F/L305I,L288A/T293K, L288A/T293K/K337A, L288A/T293K/L305V, L288A/T293K/L305I,L288A/T293R, L288A/T293R/K337A, L288A/T293R/L305V, L288A/T293R/L305I,L288A/T293Y, L288A/T293Y/K337A, L288A/T293Y/L305V, L288A/T293Y/L305I,L288A/T293F, L288A/T293F/K337A, L288A/T293F/L305V or L288A/T293F/L305I.

Embodiment 4

Factor VII polypeptide according to embodiment 2, wherein thepolypeptide has the following substitutions: L288F/T293K,L288F/T293K/K337A, L288F/T293R, L288F/T293R/K337A, L288Y/T293K,L288Y/T293K/K337A, L288Y/T293R, L288Y/T293R/K337A, L288N/T293K,L288N/T293K/K337A, L288N/T293R or L288N/T293R/K337A.

Embodiment 5

Factor VII polypeptide according to embodiment 1, wherein Q176 isreplaced by Lys (K), Arg (R), or Asn (N).

Embodiment 6

Factor VII polypeptide according to embodiment 5, wherein thepolypeptide comprises one of the following groups of substitutions:L288F/Q176K/K337A, L288Y/Q176K/K337A, L288N/Q176K/K337A orL288A/Q176K/K337A.

Embodiment 7

Factor VII polypeptide according to embodiment 1, wherein Q286 isreplaced by Asn (N).

Embodiment 8

Factor VII polypeptide comprising one or more substitutions relative tothe amino acid sequence of human Factor VII (SEQ ID NO:1), characterizedin that one substitution is where L288 is replaced by Phe (F), Tyr (Y),Asn (N) or Ala (A), with the proviso that the polypeptide does not havethe following pair of substitutions L288N/R290S or L288N/R290T.

Embodiment 9

Factor VII polypeptide according to any one of embodiments 1-2(xxx), 5and 7-8, wherein the Factor VII polypeptide further comprises one ormore of the following substitutions L305I, L305V or K337A.

Embodiment 10

Factor VII polypeptide comprising two or more substitutions relative tothe amino acid sequence of human Factor VII (SEQ ID NO:1), wherein W201is replaced by Arg (R), Met (M), or Lys (K) and wherein T293 is replacedby Lys (K), Arg (R), Tyr (Y) or Phe (F); wherein Q176 is replaced by Lys(K), Arg (R) or Asn (N); or Q286 is replaced by Asn (N).

Embodiment 10(i)

Factor VII polypeptide according to any one of embodiments 1-1(ii) or10, wherein T293 is replaced by Lys (K), Arg (R), Tyr (Y) or Phe (F) andwherein W201 is replaced by Arg (R), Met (M) or Lys (K).

Embodiment 11

Factor VII polypeptide according to embodiment 10, wherein T293 isreplaced by Lys (K), Arg (R), Tyr (Y) or Phe (F).

Embodiment 11(i)

Factor VII polypeptide according to any one of embodiments 1-2, 10 or11, wherein T293 is replaced by Lys (K), Arg (R), Tyr (Y) or Phe (F) andW201 is replaced by Arg (R), Met (M) or Lys (K).

Embodiment 11(ii)

Factor VII polypeptide according to any one of embodiments 1-2, 10 or11, wherein T293 is replaced by Lys (K) and W201 is replaced by Arg (R).

Embodiment 11(iii)

Factor VII polypeptide according to any one of embodiments 1-2, 10 or11, wherein T293 is replaced by Lys (K) and W201 is replaced by Met (M).

Embodiment 11(iv)

Factor VII polypeptide according to any one of embodiments 1-2, 10 or11, wherein T293 is replaced by Lys (K) and W201 is replaced by Lys (K).

Embodiment 11(v)

Factor VII polypeptide according to any one of embodiments 1-2, 10 or11, wherein T293 is replaced by Arg (R) and W201 is replaced by Arg (R).

Embodiment 11(vi)

Factor VII polypeptide according to any one of embodiments 1-2, 10 or11, wherein T293 is replaced by Arg (R) and W201 is replaced by Met (M).

Embodiment 11(vii)

Factor VII polypeptide according to any one of embodiments 1-2, 10 or11, wherein T293 is replaced by Arg (R) and W201 is replaced by Lys (K).

Embodiment 11(viii)

Factor VII polypeptide according to any one of embodiments 1-2, 10 or11, wherein T293 is replaced by Tyr (Y) and W201 is replaced by Arg (R).

Embodiment 11(ix)

Factor VII polypeptide according to any one of embodiments 1-2, 10 or11, wherein T293 is replaced by Tyr (Y) and W201 is replaced by Met (M).

Embodiment 11(x)

Factor VII polypeptide according to any one of embodiments 1-2, 10 or11, wherein T293 is replaced by Tyr (Y) and W201 is replaced by Lys (K).

Embodiment 11(xi)

Factor VII polypeptide according to any one of embodiments 1-2, 10 or11, wherein T293 is replaced by Phe (F) and W201 is replaced by Arg (R).

Embodiment 11(xii)

Factor VII polypeptide according to any one of embodiments 1-2, 10 or11, wherein T293 is replaced by Phe (F) and W201 is replaced by Met (M).

Embodiment 11(xiii)

Factor VII polypeptide according to any one of embodiments 1-2, 10 or11, wherein T293 is replaced by Phe (F) and W201 is replaced by Lys (K).

Embodiment 12

Factor VII polypeptide according to embodiment 11, wherein thepolypeptide comprises one of the following groups of substitutions:W201R/T293K, W201R/T293K/K337A, W201R/T293K/L305V, W201R/T293K/L305I,W201R/T293R, W201R/T293R/K337A, W201R/T293R/L305V, W201R/T293R/L305I,W201R/T293Y, W201R/T293Y/K337A, W201R/T293Y/L305V, W201R/T293Y/L305I,W201R/T293F, W201R/T293F/K337A, W201R/T293F/L305V, W201R/T293F/L305I,W201K/T293K, W201K/T293K/K337A, W201K/T293K/L305V, W201K/T293K/L305I,W201K/T293R, W201K/T293R/K337A, W201K/T293R/L305V, W201K/T293R/L305I,W201K/T293Y, W201K/T293Y/K337A, W201K/T293Y/L305V, W201K/T293Y/L305I,W201K/T293F, W201K/T293F/K337A, W201K/T293F/L305V, W201K/T293F/L305I,W201M/T293K, W201M/T293K/K337A, W201M/T293K/L305V, W201M/T293K/L305I,W201M/T293R, W201M/T293R/K337A, W201M/T293R/L305V, W201M/T293R/L305I,W201M/T293Y, W201M/T293Y/K337A, W201M/T293Y/L305V, W201M/T293Y/L305I,W201M/T293F, W201M/T293F/K337A, W201M/T293F/L305V or W201M/T293F/L305I.

Embodiment 13

Factor VII polypeptide according to embodiment 11, wherein thepolypeptide has the following substitutions: W201R/T293K,W201R/T293K/K337A, W201R/T293R, W201R/T293R/K337A, W201R/T293Y,W201R/T293F, W201K/T293K or W201M/T293K.

Embodiment 14

Factor VII polypeptide according to embodiment 10, wherein Q176 isreplaced by Lys (K), Arg (R), or Asn (N).

Embodiment 15

Factor VII polypeptide according to embodiment 14, wherein thepolypeptide comprises one of the following groups of substitutionsW201R/Q176K, W201R/Q176R, W201K/Q176K, W201K/Q176R, W201M/Q176K, orW201M/Q176R.

Embodiment 16

Factor VII polypeptide according to embodiment 10, wherein Q286 isreplaced by Asn (N).

Embodiment 17

Factor VII polypeptide according to any one of embodiments 10-11, 14,and 16, wherein the Factor VII polypeptide further comprises one or moreof the following substitutions L305I, L305V or K337A.

Embodiment 18

Factor VII polypeptide comprising one or more substitutions relative tothe amino acid sequence of human Factor VII (SEQ ID NO:1), characterizedin that one substitution is where W201 is replaced by Arg (R), Met (M),or Lys (K).

Embodiment 19

Factor VII polypeptide comprising two or more substitutions relative tothe amino acid sequence of human Factor VII (SEQ ID NO:1), wherein L288is replaced by Phe (F), Tyr (Y), Asn (N), or Ala (A); wherein W201 isreplaced by Arg (R), Met (M), or Lys (K) and, optionally, wherein T293is replaced by Lys (K), Arg (R), Tyr (Y) or Phe (F); Q176 is replaced byLys (K), Arg (R) or Asn (N); or Q286 is replaced by Asn (N).

Embodiment 20

Factor VII polypeptide according to embodiment 19, wherein thepolypeptide comprises one of the following groups of substitutionsL288F/W201K, L288F/W201R, L288F/W201M, L288N/W201K, L288N/W201R,L288N/W201M, L288Y/W201K, L288Y/W201R, L288Y/W201M, L288A/W201K,L288A/W201R, L288A/W201M, L288F/W201K/T293K, L288F/W201K/T293Y,L288F/W201R/T293K, L288F/W201R/T293Y, L288F/W201M/T293K,L288F/W201M/T293Y, L288N/W201K/T293K, L288N/W201K/T293Y,L288N/W201R/T293K, L288N/W201R/T293Y, L288N/W201M/T293K,L288N/W201M/T293Y, L288A/W201K/T293K, L288A/W201K/T293Y,L288A/W201R/T293K, L288A/W201R/T293Y, L288A/W201M/T293K,L288A/W201M/T293Y, L288Y/W201K/T293K, L288Y/W201K/T293Y,L288Y/W201R/T293K, L288Y/W201R/T293Y, L288Y/W201M/T293K orL288Y/W201M/T293Y.

Embodiment 21

Factor VII polypeptide according to any one of the precedingembodiments, wherein the Factor VII polypeptide further comprises one ormore of the following substitutions R396C, Q250C, or 407C.

Embodiment 22

Factor VII polypeptide according to any one of the previous embodiments,wherein said Factor VII polypeptide is a cleaved, two-chain Factor VIIapolypeptide.

Embodiment 22(i)

Factor VII polypeptide according to any one of the preceding embodimentscomprising two amino acid substitutions relative to the amino acidsequence of human Factor VII (SEQ ID NO:1).

Embodiment 22(ii)

Factor VII polypeptide according to any one of the preceding embodimentscomprising three amino acid substitutions relative to the amino acidsequence of human Factor VII (SEQ ID NO:1).

Embodiment 22(iii)

Factor VII polypeptide according to any one of the preceding embodimentscomprising four amino acid substitutions relative to the amino acidsequence of human Factor VII (SEQ ID NO:1).

Embodiment 22(iv)

Factor VII polypeptide according to any one of the preceding embodimentscomprising five amino acid substitutions relative to the amino acidsequence of human Factor VII (SEQ ID NO:1).

Embodiment 22(v)

Factor VII polypeptide according to any one embodiments 22(i)-(iv)comprising at the most ten amino acid substitutions relative to theamino acid sequence of human Factor VII (SEQ ID NO:1).

Embodiment 22(vi)

Factor VII polypeptide according to any one of the precedingembodiments, which has a proteolytic activity that is at least 110%,such as at least 120%, such as at least 130%, such as at least 140%,such as at least 150%, such as at least 160%, such as at least 170%,such as at least 180%, such as at least 190%, such as at least 200%,such as at least 300%, such as at least 400%, such as at least 500%,such as at least 1000%, such as at least 3000%, such as at least 5000%,such as at least 10 000%, such as at least 30 000% that of wild typehuman Factor VIIa, as measured in an in vitro proteolytic assay, in theabsence of soluble tissue factor.

Embodiment 22(vii)

Factor VII polypeptide according to any one of the precedingembodiments, which has less than 20%, such as less than 19%, such asless than 18%, such as less than 17%, such as less than 16%, such asless than 15%, such as less than 14%, such as less than 13%, such asless than 12%, such as less than 11%, such as less than 10%, such asless than 9%, such as less than 8%, such as less than 7%, such as lessthan 6%, such as less than 5% antithrombin reactivity compared to thatof wild type human Factor VIIa (SEQ ID NO: 1), as measured in anantithrombin inhibition assay, in the presence of low molecular weightheparin and the absence of soluble tissue factor.

Embodiment 23

Factor VII polypeptide according to any of the preceding embodiments,wherein the Factor VII polypeptide is coupled with at least onehalf-life extending moiety.

Embodiment 24

Factor VII polypeptide according to embodiment 23, wherein the half-lifeextending moiety is selected from biocompatible fatty acids andderivatives thereof, Hydroxy Alkyl Starch (HAS) e.g. Hydroxy EthylStarch (HES), Poly Ethylen Glycol (PEG), Poly (Glyx-Sery)n (HAP),Hyaluronic acid (HA), Heparosan polymers (HEP), Phosphorylcholine-basedpolymers (PC polymer), Fleximers, Dextran, Poly-sialic acids (PSA), Fcdomains, Transferrin, Albumin, Elastin like (ELP) peptides, XTENpolymers, PAS polymers, PA polymers, Albumin binding peptides, CTPpeptides, FcRn binding peptides and any combination thereof.

Embodiment 25

Factor VII polypeptide according to embodiment 24, wherein the half-lifeextending moiety is a heparosan polymer.

Embodiment 26

Factor VII polypeptide according to embodiment 25, wherein the heparosanpolymer has a molecular weight in a range selected from 13-65 kDa, 13-55kDa, 25-55 kDa, 25-50 kDa, 25-45 kDa, 30-45 kDa and 38-42 kDa, or amolecular weight of 40 kDa.

Embodiment 26(i)

FVII polypeptide according to any one of embodiments 25-26, comprisingthe structural fragment shown in Formula I,

wherein n is an integer from 95-115.

Embodiment 26(ii)

Factor VII polypeptide according to any one of the precedingembodiments, which has a half-life that is increased by at least 100%compared to wild type human Factor VIIa (SEQ ID NO: 1).

Embodiment 27

Factor VII polypeptide according to any of the preceding embodiments,wherein said Factor VII polypeptide is disulfide linked to tissuefactor.

Embodiment 28

Factor VII polypeptide according to any of the preceding embodiments,wherein said polypeptide has additional amino acid modifications thatincrease platelet affinity of the polypeptide.

Embodiment 29

Factor VII polypeptide according to any one of embodiments 1-22, whereinsaid polypeptide is a fusion protein comprising a Factor VII polypeptideaccording to any one of embodiments 1-22 and a fusion partnerprotein/peptide, for example an Fc domain or an albumin.

Embodiment 30

Polynucleotide that encodes a Factor VII polypeptide defined in any oneof embodiments 1-22 and 28-29.

Embodiment 31

Recombinant host cell comprising the polynucleotide according toembodiment 30.

Embodiment 32

Method for producing the Factor VII polypeptide defined in any ofembodiments 1-22 and 28-29, the method comprising cultivating a cell inan appropriate medium under conditions allowing expression of thepolynucleotide construct and recovering the resulting polypeptide fromthe medium.

Embodiment 33

Pharmaceutical composition comprising a Factor VII polypeptide asdefined in any of embodiments 1-29 and a pharmaceutically acceptablecarrier.

Embodiment 34

Method for the treatment of bleeding disorders or bleeding episodes in asubject or for the enhancement of the normal haemostatic system, themethod comprising administering a therapeutically or prophylacticallyeffective amount of a Factor VII polypeptide as defined in any ofembodiments 1-29 to a subject in need thereof.

Embodiment 35

Factor VII polypeptide as defined in any of embodiments 1-26 for use asa medicament.

Embodiment 35(i)

Factor VII polypeptide as defined in any one of embodiments 1-26 for usein the treatment of a coagulopathy.

Embodiment 36

Factor VII polypeptide according to embodiment 35(i) for use as amedicament in the treatment of haemophilia A or B.

The present invention is further illustrated by the following exampleswhich, however, are not to be construed as limiting the scope ofprotection. The features disclosed in the foregoing description and inthe following examples may, both separately and in any combinationthereof, be material for realising the invention in diverse formsthereof.

EXAMPLES Proteins

Human plasma-derived Factor X (FX) and Factor Xa (FXa) were obtainedfrom Enzyme Research Laboratories Inc. (South Bend, Ind.). Solubletissue factor 1-219 (sTF) or 1-209 were prepared according to publishedprocedures (Freskgard et al., 1996). Expression and purification ofrecombinant wild-type FVIIa was performed as described previously (Thimet al., 1988; Persson and Nielsen, 1996). Human plasma-derivedantithrombin (Baxter) was repurified by heparin sepharose chromatography(GE Healthcare) according to published procedures (Olson et al., 1993).Bovine serum albumin (BSA) was obtained from Sigma Aldrich (St. Louis,Mo.).

Example 1 FVIIa Variant Design

To design FVIIa variants with higher proteolytic activity towards FX asa substrate, a two-pronged strategy was employed. FVIIa loops and singleamino acids, around the active-site area, were selected for swapping andfor point mutagenesis, respectively, with corresponding FVII amino acidsfrom different species (FIG. 1). FVIIa proteolytic activity was measuredas outlined in example 5. Proteolytic activities for three loop-swappedFVIIa variants are shown in

Table 1 where residues at positions 287 and 289 are mutated to threonineand glutamic acid respectively while changing the amino acid at position288. It was observed that changes at position 288, while maintaining thesame amino acids at positions 287 and 289, dramatically affected theproteolytic activity. It was also observed that substituting the aminoacid at position 201 for either a leucine, carried by rat and rabbitFVII, or an arginine, carried by bovine FVII, affected the proteolyticactivity. Furthermore, it was observed that substituting the amino acidat position 337 for either a glutamine carried by horse or a less bulkyamino acid such as alanine affected the proteolytic activity (Table 1).These observations suggested that the amino acids at position 288 and201 could be involved in FX recognition and activation. Therefore,positions 288 and 201 were further investigated by saturationmutagenesis and the representative results are outlined in Table 2.

TABLE 1 Proteolytic activity of selected FVIIa variants. Results areshown in percent (%) of wild-type FVIIa. Proteolytic Proteolyticactivity + activity + PS:PC sTF + PS:PC FVIIa variant (%) (%) FVIIa 100100 FVIIa L287T L288F D289E 100 22.1 FVIIa L287T L288H D289E 27.3 4.5FVIIa L287T L288R D289E 3.2 0.7 FVIIa W201L 66.5 72.8 FVIIa W201R 404.5177.7 FVIIa K337Q 29.8 63.5 FVIIa K337A 347.3 97.7 FVIIa K337G 317.2126.1

Example 2 Cloning of FVIIa Variants

Mutations were introduced into a mammalian expression vector encodingFVII cDNA using a site directed mutagenesis PCR-based method using KODXtreme™ Hot Start DNA Polymerase from Novagen or QuickChange®Site-Directed Mutagenesis kit from Stratagene. The pQMCF expressionvector and CHOEBNALT85 from Icosagen Cell Factory (Estonia) was used asexpression system. Introduction of the desired mutations was verified byDNA sequencing (MWG Biotech. Germany).

Example 3 FVIIa Expression

The FVII variants were expressed in CHOEBNALT85 cells from Icosagen CellFactory (Estonia). Briefly, CHOEBNALT85 suspension cells weretransiently transfected by electroporation (Gene Pulse Xcell, Biorad,Copenhagen, DK). Transfected cells were selected with 700 μg/lGeneticin® (Gibco by Life Technologies), and expanded to give a total of300 ml to 10 liter supernatant. Cells were cultured in medium accordingto manufacturer's instructions supplemented with 5 mg/I Vitamin K1(Sigma-Aldrich). Depending on scale, cells were cultured in shake flasks(37° C. 5-8% CO2 and 85-125 rpm) or rocking cultivation bags (37° C. 5%CO2 and 30 rpm). Small scale supernatants were harvested bycentrifugation followed by filtration through a 0.22 μm PES filter(Corning; Fischer Scientific Biotech, Slangerup, DK) and larger volumeswere harvested by depth filtration followed by 0.22 μm absolutefiltration (3 μm Clarigard, Opticap XL10; 0.22 μm Durapore, OpticapXL10, Merck Millipore, Hellerup, DK).

Example 4 FVIIa Purification and Concentration Determination

FVII variants were purified by Gla-domain directed antibody affinitychromatography essentially as described elsewhere (Thim et al. 1988).Briefly, the protocol comprised of 3 steps. In step 1, 5 mM CaCl₂ wasadded to the conditioned medium and the sample was loaded onto theaffinity column. After extensive wash with 10 mM His, 2 M NaCl, 5 mMCaCl₂, 0.005% Tween 80, pH 6.0, bound protein was eluted with 50 mM His,15 mM EDTA, 0.005% Tween80, pH 6.0 onto (step 2) an anion exchangecolumn (Source 15Q, GE Healthcare). After wash with 20 mM HEPES, 20 mMNaCl, 0.005% Tween80, pH 8.0, bound protein was eluted with 20 mM HEPES,135 mM NaCl, 10 mM CaCl₂, 0.005% Tween80, pH 8.0 onto (step 3) aCNBr-Sepharose Fast Flow column (GE Healthcare) to which humanplasma-derived FXa had been coupled at a density of 1 mg/ml according tomanufacturer's instructions. The flow rate was optimized to ensureessentially complete activation of the purified zymogen variants to theactivated form. For FVIIa variants with enhanced activity, capable ofauto-activation in the conditioned medium or on the anion exchangecolumn, step 2 and/or step 3 were omitted to prevent proteolyticdegradation. Purified proteins were stored at −80° C. Protein qualitywas assessed by SDS-PAGE analysis and the concentration of functionalmolecules measured by active site titration or quantification of thelight chain content by rpHPLC as described below.

Measurement of FVIIa Variant Concentration by Active Site Titration

The concentration of functional molecules in the purified preparationswas determined by active site titration from the irreversible loss ofamidolytic activity upon titration with sub-stoichiometric levels ofd-Phe-Phe-Arg-chloromethyl ketone (FFR-cmk; Bachem) essentially asdescribed elsewhere (Bock P. E., 1992. J. Biol. Chem. 267. 14963-14973).Briefly, all proteins were diluted in assay buffer (50 mM HEPES, pH 7.4,100 mM NaCl, 10 mM CaCl₂. 1 mg/mL BSA, and 0.1% w/v PEG8000). A finalconcentration of 150 nM FVIIa variant was preincubated with 500 nM ofsoluble tissue factor (sTF) for 10 min followed by the addition ofFFR-cmk at final concentrations of 0-300 nM (n=2) in a total reactionvolume of 100 μL in a 96-well plate. The reactions were incubated overnight at room temperature. In an another 96-well plate, 20 μL of eachreaction was diluted 10 times in assay buffer containing 1 mM S-2288(Chromogenix, Milano, Italy). The absorbance increase was measuredcontinuously for 10 min at 405 nM in a Spectramax 190 microplatespectrophotometer equipped with SOFTmax PRO software. Amidolyticactivity was reported as the slope of the linear progress curves afterblank subtraction. Active site concentrations were determined byextrapolation, as the minimal concentration of FFR-cmk needed tocompletely abolish the amidolytic activity.

Measurement of FVIIa Variant Concentration from the Light-Chain ContentUsing Reversed-Phase HPLC—

In an alternative approach, the concentration of functional FVIIamolecules in purified preparations were determined by quantification ofthe FVIIa light chain (LC) content by reversed-phase HPLC (rpHPLC). Acalibration curve with wild-type FVIIa was prepared using FVIIaconcentrations in the range from 0 to 3 μM, while samples of unknownconcentration were prepared in estimated concentrations of 1.5 μM (n=2).All samples were reduced using a 1:1 mixture of 0.5 Mtris(2-carboxyethyl)phosphine (TCEP; Calbiochem/Merck KGaA, Darmstadt,Germany) and formic acid added to the samples to a concentration of 20%(v/v) followed by heating of samples at 70° C. for 10 min. The reducedFVIIa variants were loaded onto a C4 column (Vydac. 300 Å, particle size5 μM, 4.6 mm, 250 mm) maintained at 30° C. Mobile phases consisted of0.09% TFA in water (solvent A) and 0.085% TFA in acetonitrile (solventB). Following injection of 80 μL sample, the system was runisocratically at 25% solvent B for 4 min followed by a linear gradientfrom 25-46% B over 10 min. Peaks were detected by fluorescence usingexcitation and emission wavelengths of 280 and 348 nm, respectively.Light chain quantification was performed by peak integration andrelative amounts of FVIIa variants were calculated on basis of thewild-type FVIIa standard curve.

Example 5 Screen for Mutations Conferring Increased Activity

As outlined in example 1,

Table 1, and to evaluate the role of FVIIa amino acids at positions 201and 288; these positions were subjected to rigorous site-directedsaturation mutagenesis. In order to further identify FVIIa variantshaving enhanced proteolytic activity other amino acid positions, 305 and337, were also selected for saturation mutagenesis. Briefly, activitywas measured as the ability of each variant to proteolytically activatethe macromolecular substrate Factor X in the presence of phospholipidvesicles (In vitro proteolysis assay). Each reaction was performed inthe presence or absence of the co-factor tissue factor (sTF) to mimicthe possible TF dependent and independent mechanisms of action ofrecombinant FVIIa. Furthermore, to understand the role of thesesubstitutions towards FVIIa inhibition by antithrombin; antithrombininhibition was quantified under pseudo-first order conditions in thepresence of low molecular weight heparin to mimic the ability ofendogenous heparin-like glycosaminoglycans (GAGs) to accelerate thereaction in vivo. These results are summarized in

Table 2. As shown in FIG. 2, the measured in vitro antithrombinreactivities were found to correlate with the in vivo accumulation ofFVIIa-antithrombin complexes thus validating the predictiveness of thein vitro screening procedure.

TABLE 2 Saturation mutagenesis of selected amino acid positions. Resultsare shown in percent (%) of wild-type FVIIa Proteolytic Proteolytic ATAT activity + activity + reactivity + reactivity + PS:PC sTF + PS:PCLMWH sTF FVIIa variant (%) (%) (%) (%) FVIIa W201A 99.1 110.9 98.7 73FVIIa W201D 78 84.2 72.5 72.7 FVIIa W201E 68.4 55.9 70.6 52.1 FVIIaW201F 55 55.5 113.1 152.5 FVIIa W201H 88.8 85.8 111.4 118.9 FVIIa W201I83.5 85.1 79.3 104.5 FVIIa W201K 149 120.2 160.4 95.6 FVIIa W201L 66.572.8 96 32.7 FVIIa W201M 135.3 151.4 114.3 155.8 FVIIa W201N 79.1 56.582.9 64 FVIIa W201P 65.5 84.4 104.8 121.6 FVIIa W201Q 115.7 93.3 79.462.9 FVIIa W201R 404.5 177.7 160 59.6 FVIIa W201S 120.5 91.5 82.3 74.7FVIIa W201T 89.5 74.8 71 72 FVIIa W201V 86.1 74.3 81 86.1 FVIIa W201Y125.7 107.8 115 122.3 FVIIa L288A 206.3 75.1 112.7 87.9 FVIIa L288D 25.910.3 FVIIa L288E 91.4 37.7 77.8 91.5 FVIIa L288F 574.6 90.7 156.8 78.6FVIIa L288G 98.4 44.8 11.6 37.7 FVIIa L288K 10.3 6.2 151.4 69 FVIIaL288M 59.9 47.1 151 91.8 FVIIa L288N 279.6 44.4 85.9 21.7 FVIIa L288Q62.1 28.9 177.6 67.6 FVIIa L288S 151.2 57.4 214.7 91.7 FVIIa L288T 51.929.4 145.7 74.2 FVIIa L288V 35.2 30.8 98.9 71.5 FVIIa L288W 251.3 41.5221.1 74.5 FVIIa L288Y 530.4 73.4 152.6 89.9 FVIIa L305A 26 18.8 31 28.8FVIIa L305I 327.5 92.3 201.5 76.6 FVIIa L305T 34.8 85.9 42.5 46.1 FVIIaL305V 164.4 133.2 215.1 56.1 FVIIa K337A 347.3 97.7 157.4 128.7 FVIIaK337D 0 4.2 FVIIa K337E 20.3 39.2 3.8 29.9 FVIIa K337G 317.2 126.1 183.7208.2 FVIIa K337I 12.3 34 1.8 9.5 FVIIa K337L 8.1 15.8 1.6 13.3 FVIIaK337N 1.5 12.9 FVIIa K337Q 29.8 63.5 30.3 78.2 FVIIa K337S 49.8 112.340.4 144.3 FVIIa K337T 3.9 16 FVIIa K337V 12.4 29.9 7.9 15.5 FVIIa K337Y8.3 40.7

Amino acids including glutamine, tyrosine, methionine, lysine, andarginine at position 201 are required for gaining proteolytic activitytowards FX as substrate in presence of phospholipids. W201R provides themost gain in the proteolytic activity in presence of phopholipids and ineither absence or presence of sTF. On the other hand, amino acidsincluding phenylalanine, leucine, and asparagine decrease theproteolytic activity compared to FVIIa WT. In case of position 288,alanine, asparagine, serine, tryptophan, phenylalanine, and tyrosineprovide gain in the proteolytic activity towards FX as substrate inpresence of phospholipids. L288F and L288Y provide the most gain in theproteolytic activity in presence of phopholipids. Data presented in

Table 2 demonstrates the challenges in predicting the proteolyticactivity and antithrombin reactivity a priori. Our approach of usingsaturation mutagenesis is, therefore, justified in order to explore thefull repertoire of influence in activity that different amino acidsbring about in FVIIa variants.

Measurement of Proteolytic Activity Using Factor X as Substrate (InVitro Proteolysis Assay)—

The proteolytic activity of the FVIIa variants was estimated usingplasma-derived factor X (FX) as substrate. All proteins were diluted in50 mM HEPES pH 7.4, 100 mM NaCl, 10 mM CaCl₂, 1 mg/mL BSA, and 0.1% w/vPEG8000. Relative proteolytic activities were determined by incubating 1to 10 nM of each FVIIa conjugate with 40 nM FX in the presence of 25 μM75:25 phosphatidyl choline:phosphatidyl serine (PC:PS) phospholipids(Haematologic technologies, Vermont, USA) for 30 min at room temperaturein a total reaction volume of 100 μL in a 96-well plate (n=2). FXactivation in the presence of sTF was determined by incubating 5 μM ofeach FVIIa conjugate with 30 nM FX in the presence of 25 μM PC:PSphospholipids for 20 min at room temperature in a total reaction volumeof 100 μL (n=2). After incubation, reactions were quenched by adding 100μL of 1 mM S-2765 (Chromogenix, Milano, Italy) in stop buffer (50 mMHEPES pH 7.4, 100 mM NaCl, 80 mM EDTA). Immediately after quenching, theabsorbance increase was measured continuously at 405 nM in an Envisionmicroplate reader (PerkinElmer, Waltham, Mass.). All additions,incubations and plate movements were performed by a Hamilton MicrolabStar robot robot (Hamilton, Bonaduz, Switzeland) on line coupled to anEnvision microplate reader. Apparent catalytic rate values(k_(cat)/K_(m)) were estimated by fitting the data to a simplified formof the Michaelis Menten equation=k_(cat)*[S]*[E]/K_(m)) using linearregression since the FX substrate concentration ([S]) was below K_(m)for the activation reaction. The amount of FXa generated was estimatedfrom a standard curve prepared with human plasma-derived FXa underidentical conditions. Estimated k_(cat)/K_(m) values were reportedrelative to that of wild-type FVIIa following normalisation of themeasured rate of FXa generation according to the concentration of theFVIIa variant used. Results are given in

Table 1,

Table 2,

Table 3 and

Table 7.

Measurement of FVIIa Inhibition by Antithrombin—

A discontinuous method was used to measure the in vitro rate ofinhibition by human plasma-derived antithrombin (AT) under pseudo-firstorder conditions in the presence of low molecular weight (LMW) heparin(Calbiochem/Merck KGaA, Darmstadt, Germany). The assay was performed ina 96-well plate using a buffer containing 50 mM HEPES pH 7.4, 100 mMNaCl, 10 mM CaCl₂, 1 mg/mL BSA, and 0.1% w/v PEG8000 in a total reactionvolume of 200 μL. To a mixture of 200 nM FVIIa and 12 μM LMW heparin wasadded 5 μM antithrombin in a final reaction volume of 100 μL. Atdifferent times, the reaction was quenched by transferring 20 μL of thereaction mixture to another microtiter plate containing 180 μL of sTF(200 nM), polybrene (0.5 mg/mL; Hexadimethrine bromide, Sigma-Aldrich)and S-2288 (1 mM). Immediately after transfer at the different times,substrate cleavage was monitored at 405 nm for 10 min in an Envisionmicroplate reader. Pseudo-first order rate constants (k_(obs)) wereobtained by non-linear least-squares fitting of data to an exponentialdecay function, and the second-order rate constant (k) was obtained fromthe following relationship k=k_(obs)/[AT]. All additions, incubationsand plate movements were performed by a Hamilton Microlab Star robot(Hamilton, Bonaduz, Switzeland) on line coupled to an Envisionmicroplate reader (PerkinElmer, Waltham, Mass.). Rates of inhibitionwere reported relative to that of wild-type FVIIa. Results are given in

Table 2,

Table 3 and

Table 7.

Example 6 Combining FVIIa Mutations Conferring Increased Activity andAntithrombin Resistance

In order to design FVIIa variants with high proteolytic activity andantithrombin resistance, a selection of the identified FVIIa proteolyticactivity enhancing variants were combined with the FVIIa variants thatconfer antithrombin resistance. Specifically, FVIIa combination variantswere made with substitutions at positions 293 and 201, 288, 305, 337,176 and/or 286. Characterization of the combination FVIIa purifiedprotein preparations using the in vitro proteolysis and antithrombininhibition assays described in Example 5 are summarized in

Table 3.

Table 3 demonstrates that some combinations resulted in FVIIa variantsexhibiting a desirable high activity while at the same time having adesirable low antithrombin reactivity. For example, FVIIa variant L288FT293K displayed 600% proteolytic activity in presence of phospholipidsand just 6% antithrombin reactivity in presence of low-molecular weightheparin compared to wild-type FVIIa. Similarly, FVIIa variant L288YT293K displays 447.8% proteolytic activity in presence of phospholipidsand just 5.8% antithrombin reactivity in presence of low-molecularweight heparin compared to wild-type FVIIa. Furthermore, the W201R T293Kdisplayed 609% proteolytic activity in presence of phospholipids andjust 9% antithrombin reactivity in presence of low-molecular weightheparin compared to wild-type FVIIa.

Interestingly, combining the two FVIIa mutations L288F and K337Aprovides greatly enhanced activity with a measured 2646% increase inproteolytic activity compared to wild-type FVIIa. Upon furtherco-introduction of the mutation T293K, enhanced activity is retainedwhile a low antithrombin reactivity is achieved. This variant displays1310% proteolytic activity in presence of phospholipids and just 17%antithrombin reactivity in presence of low-molecular weight heparincompared to wild-type FVIIa.

Altogether, it can be concluded that the T293K, T293R, and T293Ymutations when combined with W201R or L288F effectively reduce theantithrombin reactivity compared to wild-type FVIIa while providinghigher proteolytic activity compared to wild-type FVIIa.

TABLE 3 Proteolytic activities and antithrombin reactivities of FVIIacombination variants. Results are shown in percent (%) of wild-typeFVIIa. Proteolytic Proteolytic AT activity + activity + sTF +reactivity + AT reactivity + PS:PC PS:PC LMWH sTF FVIIa variant (%) (%)(%) (%) FVIIa W201R T293Y 1026.9 202.6 11.1 2.3 FVIIa W201R T293R L305I1573.6 411.3 46.6 8.9 FVIIa W201R T293R 217.2 375.5 7.1 9.1 FVIIa W201RT293K L305I 1734.6 446.4 82.5 3.4 FVIIa W201R T293K 590.6 272.2 7.9 7.7FVIIa W201R L288F 2542.8 427.2 40.4 33.2 T293R FVIIa W201R L288F 1476307.2 16.7 18.2 T293K FVIIa W201M T293Y 599.7 145.4 11.6 1.7 FVIIa W201MT293R 179.2 201.7 3 6.7 FVIIa W201M T293K 146.1 173.5 3.8 5.2 FVIIaW201K T293Y 617.7 176.7 15.4 2.6 FVIIa W201K T293R 553.5 8.8 11.6 FVIIaW201K T293K 217.2 214.6 6.3 7.3 FVIIa T293Y L305V K337A 1194.6 121 62.43.3 FVIIa T293Y K337A 213 162.9 26.5 2 FVIIa T293R L305V K337A 2552.5427 37.4 6.9 FVIIa T293R L305V 1325.8 356.1 19.1 4.2 FVIIa T293R L305I711 229.7 22.1 2.6 FVIIa T293R K337A 690.2 279 10.4 13.4 FVIIa T293KL305V K337A 956.8 170.1 34.2 5.7 FVIIa T293K L305I 524 152.2 17.9 1.7FVIIa T293K K337A 773.1 264.9 7.2 6.9 FVIIa L305V T293Y 669.5 110.4 30.41.3 FVIIa L305V T293K 792.4 166.6 13.8 1.9 FVIIa L288Y T293R K337A2530.2 323.8 19.1 10.1 FVIIa L288Y T293R 1059.7 298.4 7.5 4.8 FVIIaL288Y T293K 676.5 233.7 5.4 4.5 FVIIa L288N T293Y 783.2 116.2 10.6 0.8FVIIa L288N T293R 209.5 78.7 20.7 3.5 FVIIa L288N T293K 168 69 4.4 0.9FVIIa L288F T293Y 523.9 48.1 12.2 2 FVIIa L288F T293R L305V 1784.5 101.348.6 9.1 FVIIa L288F T293R L305I 1456.4 158.2 41.4 3.5 FVIIa L288F T293RK337A 2001.9 305 21.5 20.7 FVIIa L288F T293R 259.7 110 8.3 6.7 FVIIaL288F T293K L305V 466.3 181.4 8.2 10.2 FVIIa L288F T293K L305I 2147.7147.6 33 2.8 FVIIa L288F T293K K337A 1310.7 133.6 17.1 9 FVIIa L288FT293K 600.6 210.2 6.1 4.3

Example 7 Estimation of FVIIa Potency and Plasma Level

Potencies were estimated using a commercial FVIIa specific clottingassay; STACLOT®VIIa-rTF from Diagnostica Stago. The assay is based onthe method published by J. H. Morrissey et al. Blood. 81:734-744 (1993).It measures sTF initiated FVIIa activity-dependent time to fibrin clotformation in FVII deficient plasma in the presence of phospholipids.Clotting times were measured on an ACL9000 (ILS) coagulation instrumentand results calculated using linear regression on a bilogarithmic scalebased on a FVIIa calibration curve. The same assay was used formeasurements of FVIIa clotting activity in plasma samples from animal PKstudies. The lower limit of quantification (LLOQ) in plasma wasestimated to 0.25 U/ml. Plasma activity levels were converted to nMusing the specific activity.

Example 8 Crystallographic Analysis of FVIIa Variants

To explore the mechanism by which the identified substitutions affectproteolytic activity and antithrombin recognition, crystal structures ofthe representative FVIIa variants L288Y T293K, L288F T293K, W201R T293K,W201R T293Y and L288F T293K K337A were determined.

When comparing structures on the 3-dimensional level the 1 DAN structureof wild-type (WT) FVIIa, in complex with soluble Tissue Factor, [Banner,D. W. et al, Nature, (1996), Vol. 380, 41-46] have had its heavy chainresidues of FVIIa renumbered according to the numbering scheme of SEQ IDNO: 1.

Purified H-_(D)-Phe-Phe-Arg chloromethyl ketone (FFR-cmk; Bachem,Switzerland) active-site inhibited FVIIa variants in complex withsoluble Tissue Factor (fragment 1-219) were crystallized using thehanging drop method in accordance with [Kirchhofer, D. et al, ProteinsStructure Function and Genetics, (1995), Vol. 22, pages 419-425]. Theprotein buffer solution was a mix of 10 mM Tris pH 7.5 at 25 C.°, 100 mMNaCl, 15 mM CaCl₂. Protein concentrations together with precipitantsolutions and mixing conditions for the FVIIa variants are shown in

Table 4. The hanging drop method using 24-well VDX-plates and wellsolution of 1.0 ml was utilized. The drops were set up with a mix of 1.5μl of the protein solution and 0.5 μl of the well solution. Streakseeding was used to initialize nucleation.

The cryo conditions are shown in

Table 4. The crystal was let to soak in the cryo solution for about 30seconds after which the crystal was transferred to, and flash frozen in,liquid nitrogen. Crystallographic data were processed by the XDS datareduction software [Kabsch, W., Acta Crystallographica Section DBiological Crystallography, (2010), Vol. 66, pages 125-132] usingresolution cut-off as described by Karplus et al. [Karplus, P. A. et al,Science (New York, N.Y.), (2012), Vol. 336, pages 1030-1033].

TABLE 4 Crystallization and freezing conditions for the FVIIa variants.Mixing ratio FVIIa protein:precipitant variant Protein conc. Precipitantsolution solution Cryo condition L288Y 2.5 mg/ml 0.1M Cacodylate 3:1100% TMAO T293K pH 5.1, 13% Peg (trimethylamine 8000 N-oxide) L288F 2.14mg/ml  0.1M Na-citrate 3:1 100% TMAO T293K pH 5.6, 17% Peg 3350 and 12%1- propanol W201R 1.0 mg/ml 0.1M Cacodylate 3:1 As precipitant T293K pH5.1, 13% Peg solution but with 8000 35% PEG 8000 W201R 2.93 mg/ml  0.1MCacodylate 3:1 As precipitant T293Y pH 5.1, 12% Peg solution but with35% 8000 PEG 8000 L288F 1.0 mg/ml 0.1M Na-citrate 3:1 As precipitantT293K pH 5.6, 16% Peg solution but with K337A 3350 and 12% 1- 35%PEG3350. propanol

In-house generated coordinates (unpublished) based on thecrystallographic coordinates of the 1DAN entry [Banner, D. W. et al,Nature, (1996), Vol. 380, pages 41-46] from the Protein Data Bank (PDB)[Berman, H. M. et al, Nucleic Acids Res., (2000), Vol. 28, pages235-242], were used as starting model for either molecular replacementcalculations in phenix.phaser [Mccoy, A. J. et al, J. Appl.Crystallogr., (2007), Vol. 40, pages 658-674] or straight intorefinements with the phenix.refine software [Afonine, P. V. et al, ActaCrystallogr. Sect. D-Biol. Crystallogr., (2012), Vol. 68, pages 352-367]of the PHENIX software package [Adams, P. D. et al, Acta Cryst. D,(2010), Vol. 66, pages 213-221]. Refinements were followed byinteractive model corrections in the computer graphics software COOT[Emsley, P. et al, Acta Crystallogr. Sect. D-Biol. Crystallogr., (2010),Vol. 66, pages 486-501]. Crystallographic data, refinement and modelstatistics for the 5 FVIIa variants are shown in

Table 5.

TABLE 5 Data collection, refinement and model statistics. Statistics forthe highest-resolution shell are shown in parentheses. L288F L288Y L288FW201R W201R T293K FVIIa variant T293K T293K T293K T293Y K337A Datacollection BLI911-3, BLI911-3, BLI911-3, BLI911-3, X10SA, beamlineMAX-lab MAX-lab MAX-lab MAX-lab SLS Wavelength [Å] 1.0000 1.0000 1.00001.0000 0.9999 Resolution range [Å] 35.7-2.01 29.25-2.37 29.5-1.7129.16-2.5 49.75-2.216 (2.082-2.01)  (2.455-2.37) (1.772-1.71) (2.589-2.5) (2.295-2.216) Space group P 2₁ 2₁ 2₁ P 2₁ 2₁ 2₁ P 2₁ 2₁ 2₁ P2₁ 2₁ 2₁ P 2₁ 2₁ 2₁ Unit cell [Å] 71.34 69.34 68.77 68.86 68.919 82.4681.57 78.2 81.42 81.545 123.3 125.88 172.21 125.39 125.57 Totalreflections 321033 (9043)  103617 (6525)  501348 (14728)  91616 (9007) 234709 (20365)  Unique reflections 48292 (4010)  28995 (2424)  96329(6505)  24966 (2433)  35724 (3412)  Multiplicity 6.6 (2.3) 3.6 (2.7) 5.2(2.3) 3.7 (3.7) 6.6 (6.0) Completeness [%] 98.31 (83.26) 97.71 (83.50)95.43 (63.97) 99.70 (99.43) 99.60 (96.82) Mean I/sigma(I) 9.33 (0.68)9.45 (0.79) 11.73 (0.42)  5.09 (0.54) 7.39 (0.53) Wilson B-factor [Å]22.18 40.30 24.56 25.91 45.71 R-merge 0.2439 (1.316)  0.145 (1.431)0.1141 (1.968)  0.3456 (2.778)  0.2599 (3.739)  R-meas 0.2647 0.16980.1266 0.4047 0.2822 CC1/2 0.987 (0.277) 0.992 (0.322) 0.997 (0.219)0.951 (0.11)  0.992 (0.174) CC* 0.997 (0.659) 0.998 (0.698) 0.999(0.599) 0.987 (0.446) 0.998 (0.545) Reflections used in 48286 2899196323 24966 35721 refinement Reflections used for R- 2497 1465 4773 12601853 free R-work 0.2122 (0.3487) 0.2199 (0.3698) 0.2170 (0.5007) 0.2568(0.3940) 0.2077 (0.4038) R-free 0.2561 (0.3880) 0.2755 (0.3826) 0.2556(0.5132) 0.3102 (0.4015) 0.2556 (0.4045) Number of non- 5446 4969 53824981 4566 hydrogen atoms: Total In Macromolecules 4769 4700 4836 46794355 In Ligands 81 51 87 36 39 In Waters 596 218 459 266 172 Proteinresidues 607 618 622 607 561 RMS (bonds) [Å] 0.007 0.004 0.008 0.0020.005 RMS (angles) [°] 0.88 0.64 1.05 2.73 0.75 Ramachandran 97 94 94 9495 favoured [%] Ramachandran outliers 0 0 0.99 0.51 0 [%] Clashscore1.69 1.41 3.60 2.19 1.74 Average B-factor [Å²]: 30.70 51.70 60.40 43.7061.80 Total For macromolecules 29.70 51.90 61.50 44.80 61.80 For Ligands56.10 65.60 68.50 43.60 109.30 For Solvent 35.30 44.70 46.70 23.60 50.103-Dimensional Structure Analyses

Generally there are no major differences between the wild type (WT)FVIIa molecule 1DAN structure [Banner, D. W. et al, Nature, (1996), Vol.380, pages 41-46] and those of the FVIIa variants. The overallroot-mean-square deviation (RMSD), calculated by gesamt [Krissinel, E.,Journal of Molecular Biochemistry, (2012), Vol. 1, pages 76-85] betweenthe 1DAN FVIIa heavy chain and the L288Y T293K, L288F T293K, W201RT293K, W201R T293Y and L288F T293K K337A FVIIa variants are 0.424,0.365, 0.451, 0.342 and 0.289 Å, respectively. The number of C_(α)-atompairs used in the calculations were 254, 254, 251, 254 and 254,respectively.

The W201R T293Y FVIIa Variant

Mutation FVIIa W201R:

On the detailed level the heavy chain FVIIa Arg 201 residue of thedouble mutant is situated in the “60-loop” (chymotrypsin numbering). Inthe likelihood-weighted 2mFo-DFc electron density map, at 1.0σ cut-off,there are indications of the main chain loop stretch while that cannotbe seen for the side chains of the Arg 201 residue (a Trp residue in thewild type FVIIa), together with the side chain of the residues beforeand after (Asn and Arg residues, respectively). This indicates highflexibility of those side chains. To aid in the structure interpretationa difference electron density map was calculated between observedstructure factors from the in-house wild type protein crystals and theobserved structure factors from the FVIIa double mutant [F_(obs)(WTFVIIa/sTF)−F_(obs)(FVIIa W201R T293Y/sTF)], using software from the CCP4software program package [Collaborative Computational Project, N. Actacrystallographica, Section D, Biological crystallography, 1994, 50,760-763]. Using phases from the wild type data or the double mutant dataresulted in similar difference maps. On the positive side of thedifference map the side chain of the Trp residue from the wild typeFVIIa can be clearly seen (maximum peak at 5.6σ levels using phases fromwild type data) while there is no clear indication of the Arg side chainon the negative side of the difference map. This also argues for thatthe Arg residue is more flexible than the Trp residue of the wild typeprotein. It should be noted, however, that neither the side chainsbefore nor after the Trp residue can be clearly observed in the 1 DANstructure, using a likelihood-weighted 2mFo-DFc electron density map,which is similar to the results from the FVIIa W201R T293Y/sTF crystalstructure, while the position of the Trp 201 is unmistakably seen in theFVIIa WT structure.

Regarding the main chain orientation of the loop studied, thelikelihood-weighted 2mFo-DFc electron density map and phenix.refinerefinements places the 200, 201 and 202 residues closer towards theposition of the replaced Trp side chain residue, relatively to thepublished 1DAN structure [Banner. D. W., et al., Nature, 1996, 380,41-46]. In particular residue Asn 200 has moved and its C_(α) positionis 3.1 Å away from its position in the wild type structure FIG. 3. Also,in the described [F_(obs)(WT FVIIa/sTF)−F_(obs)(FVIIa W201R T293Y/sTF)]difference map there are peaks indicating such a movement of residue Asn200. One 5.7σ positive peak close to the position of the wild type loopconformation and another 4.3σ negative peak slightly on the inside ofthe refined double mutant conformation. This supports that the mainchain has moved closer towards the position of the WT Trp side chain andrelatively more towards the center of the FVIIa heavy chain.

The structural difference seen between the wild type structure and thedouble mutated protein for the residues 200, 201 and 202 of the heavychain FVIIa probably depends on stabilization by the inward pointing Trp201 residue side chain in the WT structure that fills out a primarilyhydrophobic volume in the FVIIa protein and thereby anchors the loop inthe wild type structure. The side chain of the corresponding residue Argin FVIIa W201R T293Y does not form the same rigid structure, with atightly bound side chain, but is more flexible, and therefore notanchoring the loop in the same way as in the WT FVIIa structure.

FIG. 3 shows a stick representation of a comparison of the two crystalstructures: 1) with light-colored carbon atoms. FVIIa wild type proteinin complex with Tissue Factor, using an in-house data set from crystalsof the same type as the PDB structure 1 DAN [Banner. D. W., et al.,Nature, 1996, 380, 41-46], and 2) with dark-colored carbon atoms, theFVIIa double mutant W201R T293Y in complex with Tissue Factor. Some ofthe residues are labeled with amino acid one-letter code and ending with“-wt” or “-m” for 1) and 2), respectively. Several side chains have beentruncated (atoms outside of C_(β) have been removed) aslikelihood-weighted 2mFo-DFc electron density maps did not show anyelectron density for those side chains. For example the residues N200,R201 and R202 of the FVIIa double mutant W201R T293Y are all truncatedfor that reason. The figure was prepared by the molecular graphicssoftware PyMOL [The PyMOL Molecular Graphics System. Version 1.6.0.0Schrödinger, LLC].

Mutation FVIIa T293Y:

The heavy chain FVIIa Tyr 293 residue is situated in the activationloop 1. The likelihood-weighted 2mFo-DFc electron density map, at 1.0σcut-off, clearly show the main chain and side chain of the Tyr residuein the refined structure. The Tyr side chain atom C_(β)-C_(γ) followsthe same direction as for the C_(β)-C_(γ2) atoms in the wild type Thrresidue. The C-C_(α)-C_(β)-C_(γ) and C-C_(α)-C_(β)-C_(γ2) dihedralangles are 165 and 173° for FVIIa residue 293 of the double mutant andWT form, respectively. Thereby, the Tyr 293 residue of the double mutantdirects its side chain in the direction of the catalytic domain andtowards the binding site of the FFR-cmk bound inhibitor. The calculated[F_(obs)(WT FVIIa/sTF)−F_(obs)(FVIIa W201R T293Y/sTF)] difference mapconfirms the orientation of the Tyr side chain with a negative peak(4.7σ height) at the Tyr ring system and a positive peak (4.2σ height)at the missing Thr Oγ₁ atom.

To study the possible interactions between antithrombin and a FVIIamutated T293Y molecule a superimposition of the Factor Xa molecularcomplex with antithrombin, PDB-code 2GD4 [Johnson. D. J. D., et al.,Embo J., 2006, 25, 2029-2037], was made on the FVIIa double mutant. Themolecular graphics software PyMOL [The PyMOL Molecular Graphics System,Version 1.6.0.0 Schrödinger, LLC] was used for the superimposition ofthe FXa and FVIIa molecules and resulted in an RMSD of 0.769 Å for 1194atoms. From the riding antithrombin molecule model it is then clear thatthe Tyr 293 residue of the FVIIa W201R T293Y mutant in the theoreticallymolecular complex produced (FVIIa W201R T293Y/antithrombin III) formsspatial overlap with, in particular, residue Leu 395 but also Arg 399 ofthe antithrombin molecule FIG. 4. This is confirmed by distancecalculations, performed in the contacts software of the CCP4 programsuite, between Tyr 293 of the FVIIa double mutant and the ridingantithrombin molecule. A cut-off distance of 3.5 Å was used between theTyr 293 residue in the mutant FVIIa molecule and the antithrombinmolecule and the results are shown in

Table 6. All distances, 3.5 Å or shorter, between the residue Tyr 293 ofthe FVIIa W201R T293Y double mutant and antithrombin from theAntithrombin-S195A FXa-pentasaccharide complex, PDB:2GD4, [Johnson. D.J. D., et al., Embo J., 2006, 25, 2029-2037] after the FXa complex hasbeen superimposed on the FVIIa mutant (W201R T293Y)/sTF structure, usingthe FVIIa (W201R T293Y) and the FXa as common molecules are summarizedin

Table 6. The spatial overlap will most probably negatively influence onthe possibility for antithrombin to place its reactive center loop (RCL)into the active site of FVIIa. Thereby a T293Y mutated FVIIa moleculewill be less susceptible to inhibition by antithrombin. This is inagreement with, and gives an explanation to, what is observedexperimentally showing increased resistance to inactivation byantithrombin and prolonged half-life.

FIG. 4 is a stick representation of a theoretical model of a complexbetween antithrombin (indicated with light carbon atoms) and the FVIIaW201R T293Y double mutant (indicated with dark carbon atoms). Therelative positions of the residues Tyr 293, Gln 255, Lys 341, Gln 286 ofthe FVIIa mutant W201R T293Y, and for the antithrombin molecule residuesLeu 395, Arg 399, Glu 295, Tyr 253 and V317 are shown and labeled. Themodel was constructed based on the structures of the antithrombin/FXacomplex [Johnson. D. J. D., et al., Embo J., 2006, 25, 2029-2037], PDBcode 2GD4, where the FXa molecule, with the antithrombin let riding, hasbeen superimposed on the heavy chain of FVIIa W201R T293Y variantmolecule. Residues of FVIIa W201R T293Y and antithrombin have a prefixof “FVIIa” and “AT” respectively, followed by one-letter amino acid codeand residue number. The figure was prepared by the molecular graphicssoftware PyMOL [The PyMOL Molecular Graphics System, Version 1.6.0.0Schrödinger, LLC].

TABLE 6 All distances. 3.5 Å or shorter between the residue Tyr 293 ofthe FVIIa (W201R T293Y) double mutant and antithrombin amino acids inthe described theoretical model between the two molecules. FVIIa W201RT293Y Antithrombin Res. # Res. # and Atom Res. and Atom Distance Res.Type Chain name Type Chain name [Å] Tyr 293H N Tyr 253A OH 3.41 Tyr 293HCD1 Leu 395A CD1 3.07 Tyr 293H CD2 Tyr 253A OH 3.50 Arg 399A NH2 2.54Arg 399A CZ 3.40 Tyr 293H CE1 Leu 395A CG 2.88 Leu 395A CD1 1.89 Leu395A CD2 3.40 Tyr 293H CE2 Glu 255A OE2 3.11 Arg 399A NH2 2.24 Leu 395ACD1 2.72 Arg 399A CZ 2.90 Tyr 293H CZ Arg 399A NH2 3.26 Leu 395A CG 2.51Leu 395A CD1 1.60 Arg 399A CZ 3.43 Leu 395A CD2 2.59 Tyr 293H OH Leu395A CB 2.78 Leu 395A CG 1.53 Leu 395A CD1 1.54 Leu 395A CD2 1.26The W201R T293K FVIIa Variant

The Region Around Residue 201 of FVIIa:

On a detailed level the heavy chain FVIIa Arg 201 residue of the doublemutant is situated in the “60-loop” (chymotrypsin numbering). In thelikelihood-weighted 2mFo-DFc electron density map, at 1.0σ cut-off, themain chain loop stretch is clearly seen. The side chain of the Arg 201residue (a Trp residue in the wild type FVIIa) is also clearly observed.The outer part, the guanidinium group, of the Arg 202 residue has,however, missing electron density in the likelihood-weighted 2mFo-DFcelectron density map and at the chosen cut-off, indicating a highermobility or disorder. Regarding the main chain orientation of the loopstudied (the “60-loop”) it show transformations between the W201R T293Kand the 1DAN structure [Banner, D. W. et al, Nature, (1996), Vol. 380,pages 41-46]. After superimposing the two structures it is seen thatwhen moving along the polypeptide from residue 197 towards 203 there aredifferences in equivalent C_(α) positions by 0.64, 2.48, 3.63, 6.41,4.15 and 0.81 Å, respectively. The main chain of the loop has movedcloser towards the position of the in 1 DAN WT Trp side chain position[Banner, D. W. et al, Nature, (1996), Vol. 380, pages 41-46] and hasalso moved towards the center of the FVIIa heavy chain, the catalyticdomain. The Arg 201 residue of W201R T293K FVIIa is in the superimposedstructure placed towards the position of the replaced WT Trp side chainresidue of the published 1 DAN structure.

The structural difference seen between the wild type structure and theW201R T293K FVIIa variant of the heavy chain “60-loop” probably dependson stabilization by the inward pointing Trp 201 residue side chain inthe WT structure that fills out a primarily hydrophobic volume in theFVIIa protein and thereby anchors the loop in the wild type structure.The side chain of the corresponding, smaller, residue Arg in FVIIa W201RT293K does not anchor the loop in the same way as the Trp in the WTFVIIa structure.

The Region Around Residue 293 of FVIIa:

The heavy chain FVIIa Lys 293 residue is situated in the activationloop 1. The likelihood-weighted 2mFo-DFc electron density map, at 1.0σcut-off, clearly show the main chain and side chain of the Lys residuein the refined structure. The Lys side chain atom C_(β)-C_(γ) followsthe same direction as for the C_(β)-C_(γ2) atoms in the wild type Thrresidue. The C-C_(α)-C_(β)-C_(γ) and C-C_(α)-C_(β)-C_(γ2) dihedralangles are 169 and 173° for FVIIa residue 293 of the double mutant andWT form, respectively. The Lys 293 show a “mttt” rotamer orientation,the most common rotamer orientation for Lys [Lovell, S. C. et al,Proteins, (2000), Vol. 40, pages 389-408] as seen by the computergraphics software COOT [Emsley, P. et al, Acta Crystallogr. Sect.D-Biol. Crystallogr., (2010), Vol. 66, pages 486-501]. Moreover, the Lys293 residue N_(ζ) atom of the W201R T293K FVIIa variant makes a strong,with a distance of 2.68 Å, hydrogen bond with the residue Gln 176 O_(ε1)atom thereby stabilizing the two side chains. Compared to the WT FVIIa1DAN structure the Gln 176 residue has therefore altered its side chainconformation to optimize the hydrogen bond it makes with the Lys 293residue in the W201R T293K FVIIa variant. The rotamer goes from the“tt0°” conformation of the WT structure to a rotamer conformation whichis not among the standard conformations described in [Lovell, S. C. etal, Proteins, (2000), Vol. 40, pages 389-408]. Thereby, the Lys 293residue of the double mutant directs its side chain in the direction ofthe catalytic domain and towards the binding site of the FFR-cmk boundinhibitor and is filling out a prime site of the FVIIa active sitecleft.

The L288Y T293K FVIIa Variant

The Region Around Residue 288 of FVIIa:

The region is clearly seen in the crystal structure likelihood-weighted2mFo-DFc electron density map, at a 1.0σ cut-off. The residues in theloop following the Tyr 288 residue, residues 289 to 292 in the heavychain of the FVIIa L288Y T293K FVIIa variant shows a change in mainchain conformation with a maximum difference at residue Arg 290 wherethe C_(α) atoms differs 2.87 Å between the a superimposed molecules ofthe FVIIa L288Y T293K FVIIa variant and the WT structure of FVIIa, 1DAN[Banner, D. W. et al, Nature, (1996), Vol. 380, pages 41-46]. The C_(α)atom of the Tyr 288 residue shows a 0.80 Å difference to the equivalentatom of the Leu 288 residue in the superimposed WT FVIIa. The side chainrotamer of Tyr 288 in the FVIIa L288Y T293K FVIIa variant is “p90°”while that of the Ley side chain rotamer of the WT FVIIa 1DAN structureshow a “mt” rotamer [Lovell, S. C. et al, Proteins, (2000), Vol. 40,pages 389-408]. That results in that the two equivalent side chainspoints in different directions, seen in the difference in theC-C_(α)-C_(β)-C_(γ) dihedral angle, −69° and 157° for the L288Y T293KFVIIa variant and WT FVIIa, respectively. The hydroxyl group of the Tyr288 side chain in the L288Y T293K FVIIa variant interacts favorably withsurrounding water molecules, which are ordered in the crystal structureand the side chain folds over the loop following the Tyr 288 of theFVIIa L288Y T293K variant. The structural main chain alteration of theloop following residue 288, and the mutation of residue 288 itself,might at least partly explain the activity improvements seen of thisFVIIa variant.

The Region Around Residue 293 of FVIIa:

The 3D structure of this residue and other residues in contact with ithighly similar to what is described for the W201R T293K FVIIa variant.Therefore all conclusions drawn for the T293K mutation of that variantalso applies to the of T293K mutation of the L288Y T293K FVIIa variant.

The L288F T293K FVIIa Variant

The Region Around Residue 288 of FVIIa:

The region is clearly seen in the crystal structure likelihood-weighted2mFo-DFc electron density map, at a 1.0σ cut-off. The 3D structure ofthis region is highly similar to the L288F T293K FVIIa variant. The twovariants share same main chain orientation for example. One thing thatdiffers between the two FVIIa variants is that the Phe 288 side chainhas another preferred rotamer (“m-85°”) for its side chain, actuallypointing in the same orientation as the Leu 288 side chain of the WTFVIIa. An unusual property of the Phe 288 side chain of the L288F T293KFVIIa variant is that for Phe residues it is unusually exposed (145 Å²according to calculations by AREAIMOL of the CCP4 crystallographicprogram suite [Bailey, S., Acta Crystallogr. Sect. D-Biol. Crystallogr.,(1994), Vol. 50, pages 760-763]) to the surrounding solvent.

The Region Around Residue 293 of FVIIa:

The 3D structure of this residue and other residues in contact with itis highly similar to what is described for the W201R T293K FVIIavariant. Therefore all conclusions drawn for the T293K mutation of thatvariant also applies to the of T293K mutation of the L288F T293K FVIIavariant.

The L288F T293K K337A FVIIa Variant

The Region Around Residue 288 of FVIIa:

The region is clearly seen in the crystal structure likelihood-weighted2mFo-DFc electron density map, at a 1.0σ cut-off. The 3D structure ofthis region is highly similar to the L288F T293K FVIIa variant. The twovariants share same main chain orientation for example. One thing thatdiffers between the two FVIIa variants is that the Phe 288 side chainhas another preferred rotamer (“m-85°”) for its side chain, actuallypointing in the same orientation as the Leu 288 side chain of the WTFVIIa and the L288F T293K FVIIa variant. Therefore all conclusions drawnfor the L288F mutation of that variant also applies to the of L288Fmutation of the L288F T293K K337A FVIIa variant.

The Region Around Residue 293 of FVIIa:

The 3D structure of this residue and other residues in contact with ithighly similar to what is described for the W201R T293K FVIIa variant.Therefore all conclusions drawn for the T293K mutation of that variantalso applies to the of T293K mutation of the L288F T293K K337A FVIIavariant.

The Region Around Residue 337 of FVIIa:

The region is clearly observed in the crystal structurelikelihood-weighted 2mFo-DFc electron density map, at a 1.0σ cut-off.The 3D structure of this region is similar to the WT structure of FVIIa,1DAN [Banner, D. W. et al, Nature, (1996), Vol. 380, pages 41-46], andto the other FVIIa variants of this Example. The overall main- andside-chain orientations are close to the WT FVIIa 1 DAN structure andthe other FVIIa variant structures there are, however, smalldifferences, slightly larger than the by phenix.refinemaximum-likelihood based calculation of the coordinate error of 0.40 Åof the crystal structure. The equivalent C_(β) atoms of residue 337 are0.8 Å apart in WT structure of FVIIa, 1DAN, and the L288F T293K K337AFVIIa variant. The C_(α) atoms of the same residues are 0.4 Å apart. Theequivalent C_(α) atoms of residue 336 are 0.6 Å apart in thesuperimposed WT structure of FVIIa, 1DAN, and the L288F T293K K337AFVIIa variant. For the Phe 332 residue the side chain in shiftedapproximately 0.5 Å towards the Ala 337 residue of the L288F T293K K337AFVIIa variant compared to the WT structure of FVIIa, 1DAN [Banner, D. W.et al, Nature, (1996), Vol. 380, pages 41-46]. It can also be concludedthat the other FVIIa variants of this Example show approximately thesame deviation to the L288F T293K K337A FVIIa variant as the WTstructure of FVIIa 1 DAN does. Moreover the other FVIIa variants clustermuch closer to the WT FVIIa 1 DAN structure than the L288F T293K K337AFVIIa variant does. This might explain at least in part the alteredproperties of this variant.

Examples 9-14 Chemical Modification of FVIIa Variants Abbreviations Usedin Examples 9-14

AUS: Arthrobacter ureafaciens Sialidase

CMP-NAN: Cytidine-5′-monophosphate-N-acetyl neuraminic acid

CV: Column volume

GlcUA: Glucuronic acid

GlcNAc: N-Acetylglucosamine

GSC: 5′-Glycylsialic acid cytidine monophosphate

GSC-SH: 5′-[(4-Mercaptobutanoyl)glycyl]sialic acid cytidinemonophosphate

HEP: HEParosan polymer

HEP-GSC: GSC-functionalized heparosan polymer

HEP-[N]FVIIa: HEParosan conjugated via N-glycan to FVIIa.

HEPES: 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid

His: Histidine

PABA: p-Aminobenzamidine

ST3GalIII N-glycan specific a2,3-sialyltransferase

TCEP: tris(2-carboxyethyl)phosphine

UDP: Uridine diphosphate

Quantification Method Used in Examples 9-14:

The conjugates of the invention were analysed for purity by HPLC. HPLCwas also used to quantify amount of isolated conjugate based on a FVIIareference molecule. Samples were analysed either in non-reduced orreduced form. A Zorbax 300SB-C3 column (4.6×50 mm; 3.5 μm Agilent, Cat.No.: 865973-909) was used. Column was operated at 30° C. 5 μg sample wasinjected, and column eluted with a water (A)—acetonitrile (B) solventsystem containing 0.1% trifluoroacetic acid. The gradient program was asfollows: 0 min (25% B); 4 min (25% B); 14 min (46% B); 35 min (52% B);40 min (90% B); 40.1 min (25% B). Reduced samples were prepared byadding 10 μl TCEP/formic acid solution (70 mMtris(2-carboxyethyl)phosphine and 10% formic acid in water) to 25 μl/30μg FVIIa (or conjugate). Reactions were left for 10 minutes at 70° C.,before they were analysed on HPLC (5 μl injection).

Starting Materials Used in Examples 9-14:

HEP-Maleimide and HEP-Benzaldehyde Polymers

Maleimide and aldehyde functionalized HEP polymers of defined size isprepared by an enzymatic polymerization reaction as described in US2010/0036001. Two sugar nucleotides (UDP-GlcNAc and UDP-GlcUA) and apriming trisaccharide (GlcUA-GlcNAc-GlcUA)NH₂ for initiating thereaction is used, and polymerization is run until depletion of sugarnucleotide building blocks. The process produced HEP polymers with asingle terminal amino group. The size of HEP polymer is determined bythe sugar nucleotide to primer ratio. The terminal amine (originatingfrom the primer) is then functionalized with either a maleimidefunctionality for conjugation to GSC-SH, or a benzaldehyde functionalityfor reductive amination chemistry to the glycyl terminal of GSC.

HEP-benzaldehydes can be prepared by reacting amine functionalized HEPpolymers with a surplus of N-succinimidyl-4-formylbenzoic acid (NanoLetters (2007), 7(8), 2207-2210) in aqueous neutral solution. Thebenzaldehyde functionalized polymers may be isolated by ion-exchangechromatography, size exclusion chromatography, or HPLC. HEP-maleimidescan be prepared by reacting amine functionalized HEP polymers with asurplus of N-maleimidobutyryl-oxysuccinimide ester (GMBS; Fujiwara, K.,et al. (1988), J. Immunol. Meth. 112, 77-83).

The benzaldehyde or maleimide functionalized polymers may be isolated byion-exchange chromatography, size exclusion chromatography, or HPLC. AnyHEP polymer functionalized with a terminal primary amine functionality(HEP-NH₂) may be used in the present examples. Two options are shownbelow:

The terminal sugar residue in the non-reducing end of the polysaccharidecan be either N-acetylglucosamine or glucuronic acid (glucuronic acid isdrawn above). Typically a mixture of both sugar residues are to beexpected in the non-reducing end, if equimolar amount of UDP-GlcNAc andUDP-GlcUA has been used in the polymerization reaction.5′-Glycylsialic Acid Cytidine Monophosphate (GSC)The GSC starting material used in the current invention can besynthesised chemically (Dufner, G. Eur. J. Org. Chem. 2000, 1467-1482)or it can be obtained by chemoenzymatic routes as described inWO07056191. The GSC structure is shown below:

Example 9 Preparation of 38.8k-HEP-[N]-FVIIa L288F T293K Step 1:Synthesis of [(4-mercaptobutanoyl)glycyl]sialic acid cytidinemonophosphate (GSC-SH)

Glycyl sialic acid cytidine monophosphate (200 mg; 0.318 mmol) wasdissolved in water (2 ml), and thiobutyrolactone (325 mg; 3.18 mmol) wasadded. The two phase solution was gently mixed for 21 h at roomtemperature. The reaction mixture was then diluted with water (10 ml)and applied to a reverse phase HPLC column (C18, 50 mm×200 mm). Columnwas eluted at a flow rate of 50 ml/min with a gradient system of water(A), acetonitrile (B) and 250 mM ammonium hydrogen carbonate (C) asfollows: 0 min (A: 90%, B: 0%, C:10%); 12 min (A: 90%, B: 0%, C:10%); 48min (A: 70%, B: 20%, C:10%). Fractions (20 ml size) were collected andanalysed by LC-MS. Pure fractions were pooled, and passed slowly througha short pad of Dowex 50W×2 (100-200 mesh) resin in sodium form, beforelyophilized into dry powder. Content of title material in freeze driedpowder was then determined by HPLC using absorbance at 260 nm, andglycyl sialic acid cytidine monophosphate as reference material. For theHPLC analysis, a Waters X-Bridge phenyl column (5 μm 4.6 mm×250 mm) anda water acetonitrile system (linear gradient from 0-85% acetonitrileover 30 min containing 0.1% phosphoric acid) was used. Yield: 61.6 mg(26%). LCMS: 732.18 (MH⁺); 427.14 (MH⁺-CMP). Compound was stable forextended periods (>12 months) when stored at −80° C.

Step 2: Synthesis of 38.8 kDa HEP-GSC reagent with succinimide linkage

The HEP-GSC reagent was prepared by coupling GSC-SH([(4-mercaptobutanoyl)glycyl]sialic acid cytidine monophosphate) fromstep 1 with HEP-maleimide in a 1:1 molar ratio as follows: GSC-SH (0.68mg) dissolved in 50 mM Hepes, 100 mM NaCl, pH 7.0 (50 μl) was added 35mg of the 38.8k-HEP-maleimide dissolved in 50 mM Hepes, 100 mM NaCl, pH7.0 (1.35 ml). The clear solution was left for 2 hours at 25° C.Unreacted GSC-SH was removed by dialysis using a Slide-A-Lyzer cassette(Thermo Scientific) with a cut-off of 10 kD. The dialysis buffer was 50mM Hepes, 100 mM NaCl, 10 mM CaCl₂, pH 7.0. The reaction mixture wasdialyzed twice for 2.5 hours. The recovered material was used as such instep 4 below, assuming a quantitative reaction between GSC-SH andHEP-maleimide. The HEP-GSC reagent made by this procedure will contain aHEP polymer attached to sialic acid cytidine monophosphate via asuccinimide linkage.

Step 3: Desialylation of FVIIa L288F T293K

FVIIa L288F T293K (30 mg) was added sialidase (AUS, 100 ul, 20 U) in 10mM His, 100 mM NaCl, 60 mM CaCl₂, 10 mM PABA pH 5.9 (10 ml), and leftfor 1 hour at room temperature. The reaction mixture was then dilutedwith 50 mM HEPES, 100 mM NaCl, 1 mM EDTA, pH 7.0 (30 ml), and cooled onice. 250 mM EDTA solution (2.6 ml) was added in small portions, keepingpH at neutral by sodium hydroxide addition. The EDTA treated sample wasthen applied to a 2×5 ml HiTrap Q FF ion-exchange columns (AmershamBiosciences, GE Healthcare) equilibrated with 50 mM HEPES, 100 mM NaCl,1 mM EDTA, pH 7.0. Unbound protein was eluted with 50 mM HEPES, 100 mMNaCl, 1 mM EDTA, pH 7.0 (4 CV), followed by 50 mM HEPES, 150 mM NaCl, pH7.0 (8 CV), before eluting asialo FVIIa L288F T293K with 50 mM HEPES,100 mM NaCl, 10 mM CaCl₂, pH 7.0 (20 CV). Asialo FVIIa L288F T293K wasisolated in 50 mM Hepes, 150 mM NaCl, 10 mM CaCl₂, pH 7.0. Yield (19.15mg) was determined by quantifying the FVIIa L288F T293K light chaincontent against a FVIIa standard after tris(2-carboxyethyl)phosphinereduction using reverse phase HPLC.

Step 4: Synthesis of 38.8 kDa HEP-[N]-FVIIa L288F T293K with succinimidelinkage

To asialo FVIIa L288F T293K (19.2 mg) in 50 mM Hepes, 100 mM NaCl, 10 mMCaCl₂, 10 mM PABA, pH 7.0 (18.0 ml) was added 38.8 kDa-HEP-GSC (35 mgfrom step 2) in 50 mM Hepes, 100 mM NaCl, 10 mM CaCl₂, pH 7.0 (2.3 ml),and rat ST3GalIII enzyme (5 mg; 1.1 unit/mg) in 20 mM Hepes, 120 mMNaCl, 50% glycerol, pH 7.0 (7.2 ml). The reaction mixture was incubatedover night at 32° C. under slow rotation. The reaction mixture was thenapplied to a FVIIa specific affinity column (CV=24 ml) modified with aGla-domain specific antibody and step eluted first with 2 column volumesof buffer A (50 mM Hepes, 100 mM NaCl, 10 mM CaCl₂, pH 7.4) then 2column volumes of buffer B (50 mM Hepes, 100 mM NaCl, 10 mM EDTA, pH7.4). The method essentially follows the principle described by Thim, Let al. Biochemistry (1988) 27, 7785-779. The product with unfoldedGla-domain was collected and directly applied to a 2×5 ml HiTrap Q FFion-exchange columns (Amersham Biosciences, GE Healthcare). Column waswashed with 10 mM His, 100 mM NaCl, 0.01% Tween80, pH 7.5 (3 columnvolumes), and 10 mM His, 100 mM NaCl, 10 mM CaCl₂, 0.01% Tween80, pH 7.5(for 3.5 column volume). The pH was then lowered to 6.0 with 10 mM His,100 mM NaCl, 10 mM CaCl₂, 0.01% Tween80, pH 6.0 (3 column volumes), andthe HEPylated material eluted with 5 column volumes of a buffer mixturecomposed of 60% buffer A (10 mM His, 100 mM NaCl, 10 mM CaCl₂, 0.01%Tween80, pH 6.0) and 40% buffer B (10 mM His, 1 M NaCl, 10 mM CaCl₂,0.01% Tween80, pH 6.0). The recovered asialo FVIIa L288F T293K(unmodified) was recycled, ie. was HEPylated once more as described instep 4 and purified in the same way as just described. The combinedfractions from two hepylation runs were pooled and concentrated byultrafiltration (Millipore Amicon Ultra, cut off 10 kD).

Step 5: Capping of mono glycoconjugated heparosan 38.8k-HEP-[N]-FVIIaL288F T293K

Non-sialylated N-glycanes of 38.8k-HEP-[N]-FVIIa L288F T293K werefinally capped (i.e. sialylated) with ST3GalIII enzyme and CMP-NAN asfollows: 38.8k-HEP-[N]-FVIIa L288F T293K (5.85 mg) was incubated withST3GalIII (0.18 mg/ml); CMP-NAN (4.98 mM) in 8.4 ml of 10 mM His, 100 mMNaCl, 10 mM CaCl₂, 0.01% Tween80, pH 6.0 for 1 h at 32° C. The reactionmixture was then applied to a FVIIa specific affinity column modifiedwith a Gla-domain specific antibody and step eluted first with 2 columnvolumes of buffer A (50 mM Hepes, 100 mM NaCl, 10 mM CaCl₂, pH 7.4) then2 column volumes of buffer B (50 mM Hepes, 100 mM NaCl, 10 mM EDTA, pH7.4). The pooled fractions containing 38.8k-HEP-[N]-FVIIa L288F T293Kwere combined and dialyzed using a Slide-A-Lyzer cassette (ThermoScientific) with a cut-off of 10 kD. The dialysis buffer was 10 mM His,100 mM NaCl, 10 mM CaCl₂, 0.01% Tween80, pH 6.0. The proteinconcentration was determined by light-chain HPLC analysis after TCEPreduction. The overall yield of 38.8k-HEP-[N]-FVIIa L288F T293K was 2.46mg (13%).

Example 10 Preparation of 41.5 kDa-HEP-[N]-FVIIa L288F T293K K337A Step1: Synthesis of 41.5 kDa HEP-GSC reagent with 4-methylbenzoyl linkage

Glycyl sialic acid cytidine monophosphate (GSC) (20 mg; 32 μmol) in 5.0ml 50 mM Hepes, 100 mM NaCl, 10 mM CaCl₂ buffer, pH 7.0 was added 41.5kDa HEP-benzaldehyde (99.7 mg; 2.5 μmol). The mixture was gently rotateduntil all HEP-benzaldehyde had dissolved. During the following 2 hours,a 1M solution of sodium cyanoborohydride in MilliQ water was added inportions (5×50 μl), to reach a final concentration of 48 mM. Reactionmixture was left at room temperature overnight. Excess of GSC was thenremoved by dialysis as follows: the total reaction volume (5250 μl) wastransferred to a dialysis cassette (Slide-A-Lyzer Dialysis Cassette,Thermo Scientific Prod No. 66810 with cut-off 10 kDa capacity: 3-12 ml).Solution was dialysed for 2 hours against 2000 ml of 25 mM Hepes buffer(pH 7.2) and once more for 17 h against 2000 ml of 25 mM Hepes buffer(pH 7.2). Complete removal of excess GSC from inner chamber was verifiedby HPLC on Waters X-Bridge phenyl column (4.6 mm×250 mm, 5 μm) and awater acetonitrile system (linear gradient from 0-85% acetonitrile over30 min containing 0.1% phosphoric acid) using GSC as reference. Innerchamber material was collected and freeze dried to give 41.5 kDa HEP-GSCas white powder. The HEP-GSC reagent was analysed by NMR and on SECchromatography. The HEP-GSC reagent made by this procedure contains aHEP polymer attached to sialic acid cytidine monophosphate via a4-methylbenzoyl linkage.

Step 2: Desialylation of FVIIa L288F T293K K337A

To a solution of FVIIa L288F T293K K337A (43.5 mg) in 21 ml of 10 mMHis, 100 mM NaCl, 60 mM CaCl₂, 10 mM PABA, pH 6.7 buffer was addedsialidase (Arthrobacter ureafaciens, 9 units/ml). The reaction mixturewas incubated for 1 hour at room temperature. The reaction mixture wasthen cooled on ice and added 14 ml of 10 mM His, 100 mM NaCl pH 7.7. 50ml of a 100 mM EDTA solution was then added while maintaining neutralpH. The reaction mixture was then diluted with 50 ml of MilliQ water,and applied to 4×5 ml interconnected HiTrap Q FF ion-exchange columns(Amersham Biosciences, GE Healthcare) equilibrated in 50 mM HEPES, 50 mMNaCl, pH 7.0. Unbound protein including sialidase was eluted with 5 CVof 50 mM HEPES, 150 mM NaCl, pH 7.0. Desialylated protein was elutedwith 12 CV of 50 mM HEPES, 150 mM NaCl, 30 mM CaCl2, pH 7.0. Fractionscontaining protein were combined and added 0.5M PABA to reach a finalconcentration of 10 mM. Protein yield was determined by quantifying theFVIIa L288F T293K K337A light chain against a FVIIa standard aftertris(2-carboxyethyl)phosphine reduction using reverse phase HPLC asdescribed above. 32.5 mg asialo FVIIa L288F T293K K337A (2.83 mg/ml) wasin this way isolated in 11.5 ml of 50 mM Hepes, 150 mM NaCl, 30 mMCaCl₂, 10 mM PABA, pH 7.0.

To asialo FVIIa L288F T293K K337A (16.3 mg) in 5.75 ml of 50 mM Hepes,150 mM NaCl, 30 mM CaCl₂, 10 mM PABA, pH 7.0 was added 41.5 kDa HEP-GSC(3 equivalents, 41.5 mg) and rat ST3GalIII enzyme (2.93 mg; 1.1 unit/mg)in 4.2 ml of 20 mM Hepes, 120 mM NaCl, 50% glycerol, pH 7.0. PABAconcentration was then adjusted to 10 mM with a 0.5M aqueous PABAsolution, and pH was adjusted to 6.7 with 1N NaOH. The reaction mixturewas incubated overnight at 32° C. under slow stirring. A solution 157 mMCMP-NAN in 50 mM Hepes, 150 mM NaCl, 10 mM CaCl2, pH 7.0 (356 μl) wasthen added, and the reaction was incubated at 32° C. for an additionalhour. HPLC analysis showed a product distribution containing un-reactedFVIIa L288F T293K K337A (68%), mono HEPylated FVIIa (25%) and variouspolyHEPylated forms (7%).

The entire reaction mixture was then applied to a FVIIa specificaffinity column (CV=24 ml) modified with a Gla-domain specific antibodyand step eluted first with 2 column volumes of buffer A (50 mM Hepes,100 mM NaCl, 10 mM CaCl₂, pH 7.4) then 2 column volumes of buffer B (50mM Hepes, 100 mM NaCl, 10 mM EDTA, pH 7.4). The method essentiallyfollows the principle described by Thim, L et al. Biochemistry (1988)27, 7785-779.

The products with unfolded Gla-domain was collected and directly appliedto 3×5 ml interconnected HiTrap Q FF ion-exchange columns (AmershamBiosciences, GE Healthcare) equilibrated with a buffer containing 10 mMHis, 100 mM NaCl, pH 6.0. The column was washed with 4 column volumes of10 mM His, 100 mM NaCl, pH 6.0. Unmodified FVIIa L288F T293K K337A waseluted with 12 CV of 10 mM His, 100 mM NaCl, 10 mM CaCl₂, pH 6.0(elution buffer A). 41.5 kDa-HEP-[N]-FVIIa L288F T293K K337A was theneluted with 15 CV of 10 mM His, 325 mM NaCl, 10 mM CaCl₂, pH 6.0. Purefractions were combined, and protein concentration was determined byHPLC quantification method previously described. 3.42 mg (21%) pure 41.5kDa-HEP-[N]-FVIIa L288F T293K K337A was isolated. The combined fractionscontaining 41.5 kDa-HEP-[N]-FVIIa L288F T293K K337A was finally dialyzedagainst 10 mM His, 100 mM NaCl, 10 mM CaCl₂, pH 6.0 using aSlide-A-Lyzer cassette (Thermo Scientific) with a cut-off of 10 kD, andconcentration adjusted to (0.40 mg/ml) by adding dialysis buffer.

Example 11 Preparation of 41.5 kDa HEP-[N]-FVIIa W201 T293K

This material was prepared essentially as described in example 10. FVIIaW201R T293K (40 mg) was initially desialylated and asialo FVIIa W201RT293K (27.2 mg) was isolated by the Gla-specific ion-exchange method.The desialylated analogue was then incubated with 41.5 kDa HEP-GSC(produced as described in example 10) and ST3GalIII. The conjugationproduct was then isolated by ion-exchange chromatography. Final bufferexchange afforded 2.9 mg (7.5%) of 41.5 kDa HEP-[N]-FVIIa W201 T293K in10 mM His, 100 mM NaCl, 10 mM CaCl₂, pH 6.0.

Example 12 Preparation of 41.5 kDa HEP-[N]-FVIIa L288Y T293K

This material was prepared essentially as described in example 10. FVIIaL288Y T293K (19.9 mg) was initially desialylated and asialo FVIIa L288YT293K (16.9 mg) was isolated by the Gla-specific ion-exchange method.The desialylated analogue was then incubated with 41.5 kDa HEP-GSC(produced as described in example 10) and ST3GalIII. The conjugationproduct was then isolated by ion-exchange chromatography. Final bufferexchange afforded 1.95 mg (11.5%) of 41.5 kDa HEP-[N]-FVIIa L288Y T293Kin 10 mM His, 100 mM NaCl, 10 mM CaCl₂, pH 6.0.

Example 13 Preparation of 41.5 kDa HEP-[N]-FVIIa L288Y T293R

This material was prepared essentially as described in example 10. Afterdesilylation asialo FVIIa L288Y T293R (30 mg) was reacted with 41.5 kDaHEP-GSC (produced as described in example 10) and ST3GalIII. Theconjugation product was then isolated by ion-exchange chromatography.Final buffer exchange afforded 4.33 mg (14.4%) of 41.5 kDa HEP-[N]-FVIIaL288Y T293R was obtained in 10 mM His, 100 mM NaCl, 10 mM CaCl₂, pH 6.0.

Example 14 Preparation of 41.5 kDa HEP-[N]-FVIIa T293K K337A

This material was prepared essentially as described in example 10. Afterdesilylation asialo FVIIa L288Y T293R (8 mg) was reacted with 41.5 kDaHEP-GSC (produced as described in example 10) and ST3GalIII. Theconjugation product was isolated by ion-exchange chromatography. Finalbuffer exchange afforded 1.72 mg (15%) of 41.5 kDa HEP-[N]-FVIIa T293KK337A in 10 mM His, 100 mM NaCl, 10 mM CaCl₂, pH 6.0.

Example 15 Functional Properties of Modified Combination FVIIa Variants

FVIIa combination variants glycoconjugated to either PEG or heparosan(HEP), as described in examples 9-14, were characterized for proteolyticactivity and antithrombin reactivity as described in example 5. Resultsare summarized in

Table 7. These data show that chemical modifications of FVIIa, in thecases with PEG or HEP, decreases FVIIa variant proteolytic activity butfor some variants allows to retain higher than wild-type FVIIaproteolytic activity and further allows to retain antithrombinresistance.

TABLE 7 Functional properties of modified FVIIa variants. Results areshown in percent (%) of wild-type FVIIa. Proteolytic Proteolyticactivity + ATIII ATIII activity + sTF + Inhibition + Inhibition + PS:PCPS:PC LMWH sTF IUPAC name (% WT) (% WT) (% WT) (% WT) 40k-PEG-[N]-FVIIaW201R T293Y 266.9 88.8 5.9 1.9 40k-HEP-[N]-FVIIa W201R T293K 155.3 236.70.2 1.2 40k-HEP-[N]-FVIIa L288Y T293R 407 200.3 5.2 4.440k-HEP-[N]-FVIIa L288Y T293K 264.9 147.4 3.7 2.9 40k-HEP-[N]-FVIIaL288F T293K 555.5 115 K337A 40K-HEP-[N]-FVIIa L288F T293K 279.3 116.44.4 3.6

Example 16 Evaluation in Haemophilia A-Like Whole BloodThrombelastography

Thrombelastography (TEG) assay was chosen to evaluate activity of FVIIavariants in a heamophilia A-like whole blood by comparison to FVIIa. TEGassay provides a profile of the entire coagulation process—initiation,propagation and final clot strength measurements. Apart from thepossible influence of shear forces in flowing blood and the vasculature,TEG assay mimics the in vivo conditions of coagulation as the methodmeasures the visco elastic properties of clotting whole blood (Viuff, E.et al, Thrombosis Research, (2010), Vol. 126, pages 144-149). Each TEGassay was initiated by using kaolin and TEG parameters clotting time (R)and maximum thrombus generation rate (MTG) were recorded and reported in

Table 8. The clotting time (R) denotes the latency time from placingblood in the sample cup until the clot starts to form (2 mm amplitude);whereas, the maximum thrombus generation (MTG) denotes the velocity ofclot formation. The clotting time (R) in seconds is determined usingstandard TEG software; whereas, MTG is calculated as the firstderivative of the TEG track multiplied with 100 (100×mm/second).

Blood samples were obtained from normal, healthy donors who were membersof the Danish National Corps of Voluntary Blood Donors and met thecriteria for blood donation. Blood was sampled in 3.2% citratevacutainers (Vacuette ref. 455322, Greiner bio-one, Lot A020601 2007-02)and assayed within 60 minutes. Haemophilia A-like blood was preparedfrom normal human whole blood by addition of anti-human FVIII (Sheepanti-human FVIII, Lot AA11-01, Haematologic Technologies, VT, USA)antibody to a final concentration of 10 Bethesda Units (BU) per ml(final 0.1 mg/ml) and rotated gently at 2 rpm/min for 30 min at roomtemperature. The test compounds were added at 0.1, 1, 10 and 25 nM finalconcentrations besides FVIIa L288Y T293K that was tested in 0.069, 0.69,6.9, 17.3 nM and FVIIa W201R T293K that was tested in 0.076, 0.76, 7.6,19.1 nM.

Data from the kaolin-induced TEG showed that all compoundsdose-dependently decreased clotting time (R-time) and increases maximumthrombus generation (MTG) in haemophilia A-like blood (Table 8).

All 40k-HEP-[N]FVIIa-variants showed shorter or similar clot timecompared with FVIIa when evaluated in the highest test concentration.Also maximum thrombus generation of the variants was as similar orincreased relative to FVIIa. Moreover, the data showed that40k-HEPylation of FVIIa variants reduced the activity of the40k-HEPylated compounds when compared to their corresponding FVIIavariants (with no 40k-EPylation).

Table 8 shows the R-time (clot time) and MTG (maximum thrombusgeneration) of test compounds in kaolin-induced TEG in Haemophila A-likewhole blood. The highest concentration of test compound was 25 nMbesides FVIIa L288Y T293K that was tested in 17.3 nM and FVIIa W201RT293K that was tested in 19.1 nM. FVIIa, 40k-PEG-[N]-FVIIa and40k-HEP-[N]-FVIIa was tested in four individual donors (n=4) whereas theremaining compounds were tested in two individual donors (n=2). Data insquare brackets indicate the range for the parameter from the fourindividual donors.

TABLE 8 Thromboelastography parameters for selected FVIIa variants inHaemophilia A-like whole blood. R-time Test compound mean MTG (athighest concentration) (sec) (×100 mm/sec) FVIIa 526 21.6 [480; 580][19.4; 26.1] 40k-PEG-[N]-FVIIa 753 17.9 [680; 835] [16.1; 19.9]40k-HEP-[N]-FVIIa 668 19.0 [580; 835] [15.8; 22.4] FVIIa L288Y T293K 34524.5 [320; 370] [24.1; 24.8] 40k-HEP-[N]-FVIIa L288Y T293K 485 21.5[465; 505] [20.3; 22.7] FVIIa L288Y T293R 313 25.5 [305; 320] [24.3;26.8] 40k-HEP-[N]-FVIIa L288Y T293R 398 23.4 [370; 425] [23.1; 23.7]FVIIa L288F T293K 400 25.9 [375; 425] [23.3; 28.5] 40k-HEP-[N]-FVIIaL288F T293K 498 21.6 [425; 570] [19.0; 24.1] FVIIa L288F T293K K337A 28027.2 [255; 305] [26.9; 27.6] 40k-HEP-[N]-FVIIa L288F T293K 348 25.7K337A [335; 360] [23.4; 28.0] FVIIa W201R T293K 390 25.2 [335; 445][22.4; 28.1] 40k-HEP-[N]-FVIIa W201R T293K 345 25.7 [295; 395] [23.7;27; 7] FVIIa T293K K337A 355 25.5 [350-360] [24.3; 26.6]40k-HEP-[N]-FVIIa T293K K337A 423 22.2 [420; 425] [21.7; 22.8]

Example 17 Assessment of PK in Rat

A pharmacokinetic analysis of identified FVIIa variants in an unmodifiedform or glycoconjugated with either PEG or heparosan (HEP) was performedin rats to assess their effect on the in vivo survival of FVIIa. SpragueDawley rats (three per group) were dosed intravenously. Stabylite™(TriniLize Stabylite Tubes; Tcoag Ireland Ltd, Ireland) stabilizedplasma samples were collected as full profiles at appropriate timepoints and frozen until further analysis. Plasma samples were analysedfor clot activity (as described in Example 7) and by an ELISAquantifying FVIIa-antithrombin complexes. Pharmacokinetic analysis wascarried out by non-compartmental methods using Phoenix WinNonlin 6.0(Pharsight Corporation). The following parameters were estimated: Cmax(maximum concentration) of FVIIa-antithrombin complex, T½ (thefunctional terminal half-life) and MRT (the functional mean residencetime) for clot activity.

Briefly, FVIIa-antithrombin complexes were measured by use of an enzymeimmunoassay (EIA). A monoclonal anti-FVIIa antibody that binds to theN-terminal of the EGF-domain and does not block antithrombin binding isused for capture of the complex (Dako Denmark A/S, Glostrup; productcode O9572). A polyclonal anti-human AT antibody peroxidase conjugatewas used for detection (Siemens Healthcare Diagnostics ApS,Ballerup/Denmark; product code OWMG15). A preformed purified complex ofhuman wild-type or variant FVIIa and plasma-derived human antithrombinwas used as standard to construct EIA calibration curves. Plasma sampleswere diluted and analysed and mean concentration of duplicatemeasurements calculated. The intra-assay precision of the EIA wasbetween 1-8%.

Pharmacokinetic estimated parameters are listed in Table 9. Relative towild-type FVIIa, the tested variants exhibited reduced accumulation ofFVIIa-antithrombin complexes (Rat AT complex) with plasma levelsapproaching the detection level. Furthermore, a significantly prolongedfunctional half-life of 40k-HEP-[N]-FVIIa L288F T293K (18.4 hrs in rat)was observed compared to 40k-PEG-[N]-FVIIa (7.4 hrs in rat).

In conclusion, the presence of Lys at position 293 increases the T½ inrat and reduces the FVIIa-antithrombin complex formation. Furthermore,introduction of glycoconjugated heparason substantially improves the T½in rat.

TABLE 9 Pharmacokinetic estimated parameters for selected FVIIa variantsin rat. Rat AT complex T½ in rat MRT in rat Cmax/dose FVIIa variant(hrs) (hrs) (kg/l) FVIIa  0.8 ± 0.01  1.1 ± 0.03  0.6 ± 0.0840k-PEG-[N]-FVIIa  7.4 ± 0.20  8.3 ± 0.30  0.7 ± 0.05 40k-HEP-[N]-FVIIaL288Y T293K 15.9 ± 0.5 20.6 ± 1.0 0.04 ± 0.004 40k-HEP-[N]-FVIIa L288YT293R 11.5 ± 0.5 13.9 ± 0.6 0.05 ± 0.004 FVIIa L288F T293K  1.2 ± 0.02 1.6 ± 0.30 0.07 ± 0.01  40k-HEP-[N]-FVIIa L288F T293K 18.4 ± 3.4 20.5 ±.6 0.04 ± 0.000 40k-HEP-[N]-FVIIa W201R T293K 21.1 ± 0.5 24.8 ± 0.8 Notmeasured 40k-HEP-[N]-FVIIa L288F T293K 11.0 ± 1.6 11.5 ± 0.8 0.14 ±0.01  K337A 40k-HEP-[N]-FVIIa T293K K337A 12.4 ± 0.1 15.4 ± 0.3 0.05 ±0.004 T½: Terminal half-life of the active molecule following IVadministration MRT: Mean residence time of the active molecule followingIV administration. AT complex Cmax/dose: Maximum measured level ofcompound-antithrombin complex divided by the dose.

Example 18 Liquid Formulation of FVIIa L288Y T293K Through Active SiteStabilization

The stability of FVIIa in solution is limited by a number ofmodifications to the polypeptide chain occurring as a result ofautoproteolysis, oxidation, deamidation, isomerization, etc. Previousstudies have identified three sites on the heavy chain that aresusceptible to autoproteolytic attack; these are Arg290-Gly291,Arg315-Lys316, and Lys316-Val317 (Nicolaisen et al., FEBS, 1993,317:245-249). Calcium-free conditions further promote proteolyticrelease of the first 38 residues of the light chain encompassing theγ-carboxyglutamic acid (Gla) domain.

Here we have used the small molecule, PCI-27483-S(2-{2-[5-(6-Carbamimidoyl-1H-benzoimidazol-2-yl)-6,2′-dihydroxy-5′-sulfamoyl-biphenyl-3-yl]-acetylamino}-succinicacid), which stabilize the active site of FVIIa through non-covalentinteractions and to prevent autoproteolysis of the heavy chain in aliquid formulation (See WO2014/057069 for further details onPCI-27483-S).

Quantification of heavy chain cleavage has been assessed by analysis ofreduced FVIIa L288Y T293K with reversed phase HPLC (RP-HPLC). The assaysolution was reduced in 127 mM dithiothreitol (DTT) and 3M guanidiniumhydrochloride, which were incubated for 60° C. in 15 min, followed bythe addition of 1 μL concentrated acetic acid (per 50 μL of originalassay solution) and cooling to 25° C. 25 μg reduced FVIIa L288Y T293Kwere then injected on a ACE 3 μM C4 column (300 Å, 4.6×100 mm; AdvancedChromatography Technologies LTD, Scotland) which were temperatureequilibrated at 40° C. The protein fragments were separated with alinear gradient having a mobile phase A consisting of 0.05%trifluoroacetic acid (TFA) in water and going from 35-80% of mobilephase B consisting of 0.045% TFA in 80% acetonitrile. The gradient timewas 30 min with a flow rate of 0.7 mL/min and peak elution were detectedwith absorbance at 215 nm.

The formulation of FVIIa L288Y T293K were made up by 1.47 mg/mL CaCl₂,7.5 mg/mL NaCl, 1.55 mg/mL L-Histidine, 1.32 mg/mL Glycylglycine, 0.5mg/mL L-Methionine, 0.07 mg/mL Polysorbate 80, 0.021 mg/ml PCI-27483-S,a protein concentration of 1 mg/ml (i.e. a protein inhibitor mole ratioof 1:1.75) and a final pH of 6.8. The solution was incubated for 1 monthat 30° C. under quiescent conditions and away from light. As seen in

Table 10 the presence of PCI-27483-S, led to near-complete inhibition ofheavy chain cleavage of FVIIa L288Y T293K; whereas, no addition ofPCI-27483-S led to a prominent increase in the cleavage at the positions315-316 and 290-291.

TABLE 10 Percentage increase of the peak areas relative to day zero ofheavy chain fragments corresponding to two different cleavage sites asdetermined in the RP-HPLC chromatograms upon 28 days of incubation withand without PCI-27483-S inhibitor. Cleavage site With PCI-27483-SWithout PCI-27483-S 315-316 20% 287% 290-291 17% 675%

Example 19 In Silico Assessment of Immunogenicity Risk

The in-silico study investigated whether the novel peptides sequencesthat results from protein engineering to generate FVIIa analogues couldresult in peptide sequences capable of binding to majorhistocompatibility complex class II (MHC-II), also known as HLA-II inhumans. Such binding is pre-requisite for the presence of T-cellepitopes. The peptide/HLA-II binding prediction software used in thisstudy was based on two algorithms, NetMHCIIpan 2.1 (Nielsen et al.2010), performing HLA-DR predictions, and NetMHCII 2.2 (Nielsen et al.2009) performing HLA-DP/DQ predictions.

An Immunogenicity Risk Score (IRS) was calculated in order to be able tocompare the different FVIIa analogues with regard to immunogenicity riskpotential. The calculation was performed as follows: FVIIa wild-type wasused as reference and only predicted 15mers not in the reference (FVIIawild-type) which had a predicted Rank of 10 or less were included in theanalysis. The HLA-II alleles were classified into three classes: Class 1(Rank<=1) with weight of 2. Class 2 (1>Rank<=3) with weight of 0.5 andClass 3 (3>Rank<=10) with weight of 0.2. The class weight (2. 0.5 or0.2) was multiplied by the allele frequency (for each population) togive the IRS. The sum of IRS was calculated for each population and eachHLA-II (DRB1, DP and DQ).

The calculated risk scores for select single and combination variantsare given in

Table 11. Particularly favourable combinations include L288F/T293K,L288F/T293K/K337A, L288Y/T293K and L288Y/T293K/K337A which at the sametime exhibit a high proteolytic activity as well as reducedsusceptibility to inhibition by antithrombin.

TABLE 11 Calculated risk scores for select single and combination FVIIavariants FVIIa variant Risk score FVIIa L288F T293K 0.10 FVIIa L288FT293K K337A 0.10 FVIIa L288F T293K L305I 0.40 FVIIa L288F T293K L305V0.35 FVIIa L288F T293R 0.12 FVIIa L288F T293R K337A 0.12 FVIIa L288FT293R L305I 0.42 FVIIa L288F T293R L305V 0.37 FVIIa L288F T293Y 0.32FVIIa L288F T293Y K337A 0.32 FVIIa L288N T293K 0.06 FVIIa L288N T293R0.08 FVIIa L288N T293Y 0.26 FVIIa L288Y T293K 0.06 FVIIa L288Y T293KK337A 0.06 FVIIa L288Y T293R 0.09 FVIIa L288Y T293R K337A 0.09 FVIIaL305V T293K 0.28 FVIIa L305V T293Y 0.49 FVIIa T293K K337A 0.02 FVIIaT293K L305I 0.32 FVIIa T293K L305V K337A 0.28 FVIIa T293R K337A 0.06FVIIa T293R L305I 0.36 FVIIa T293R L305V 0.32 FVIIa T293R L305V K337A0.32 FVIIa T293Y K337A 0.23 FVIIa T293Y L305V K337A 0.49 FVIIa W201KT293K 0.19 FVIIa W201K T293R 0.23 FVIIa W201K T293Y 0.39 FVIIa W201MT293K 1.03 FVIIa W201M T293R 1.06 FVIIa W201M T293Y 1.23 FVIIa W201RL288F T293K 0.32 FVIIa W201R L288F T293R 0.34 FVIIa W201R T293K 0.25FVIIa W201R T293K L305I 0.55 FVIIa W201R T293R 0.29 FVIIa W201R T293RL305I 0.58 FVIIa W201R T293Y 0.45

The invention claimed is:
 1. A Factor VII polypeptide comprising two ormore substitutions relative to the amino acid sequence of human FactorVII (SEQ ID NO: 1), wherein T293 is replaced by Lys (K), Arg (R), Tyr(Y) or Phe (F); and L288 is replaced by Phe (F), Tyr (Y), Asn (N), Ala(A) or Trp (W) and/or W201 is replaced by Arg (R), Met (M) or Lys (K)and/or K337 is replaced by Ala (A) or Gly (G).
 2. The Factor VIIpolypeptide according to claim 1, wherein T293 is replaced by Lys (K),Arg (R), Tyr (Y) or Phe (F); and L288 is replaced by Phe (F), Tyr (Y),Asn (N), Ala (A) or Trp (W).
 3. The Factor VII polypeptide according toclaim 1, wherein T293 is replaced by Lys (K) and L288 is replaced by Phe(F).
 4. The Factor VII polypeptide according to claim 1, wherein T293 isreplaced by Lys (K) and L288 is replaced by Tyr (Y).
 5. The Factor VIIpolypeptide according to claim 1, wherein T293 is replaced by Arg (R)and L288 is replaced by Phe (F).
 6. The Factor VII polypeptide accordingto claim 1, wherein T293 is replaced by Arg (R) and L288 is replaced byTyr (Y).
 7. The Factor VII polypeptide according to claim 1, whereinK337 is replaced by Ala (A).
 8. The Factor VII polypeptide according toclaim 1, wherein T293 is replaced by Lys (K), Arg (R), Tyr (Y) or Phe(F); and W201 is replaced by Arg (R), Met (M), or Lys (K).
 9. The FactorVII polypeptide according to claim 8, wherein T293 is replaced by Lys(K) and W201 is replaced by Arg (R).
 10. The Factor VII polypeptideaccording to claim 1, wherein the Factor VII polypeptide is coupled withat least one half-life extending moiety.
 11. The Factor VII polypeptideaccording to claim 10, wherein the half-life extending moiety isselected from biocompatible fatty acids and derivatives thereof, hydroxyalkyl starch (HAS), polyethylene glycol (PEG), hyaluronic acid (HA),heparosan polymers (HEP), phosphorylcholine-based polymers (PC polymer),fleximers, dextran, poly-sialic acids (PSA), Fc domains, transferrin,albumin, elastin like peptides (ELP), XTEN polymers, PAS (Pro, Ala, Ser)polymers, PA (Pro, Ala) polymers, albumin binding peptides, CTP (carboxyterminal peptide), FcRn binding peptides and any combination thereof.12. The Factor VII polypeptide according to claim 11, wherein thehalf-life extending moiety is a heparosan polymer.
 13. The FVIIpolypeptide according to claim 1, which has a proteolytic activity thatis at least 110% that of wild type human Factor VIIa, as measured in anin vitro proteolytic assay, in the absence of soluble tissue factor; andwhich has less than 20% antithrombin reactivity compared to wild typehuman Factor VIIa, as measured in an antithrombin inhibition assay, inthe presence of low molecular weight heparin and the absence of solubletissue factor.
 14. A pharmaceutical composition comprising the FactorVII polypeptide of claim 1 and a pharmaceutically acceptable carrier.15. A method of treating a coagulopathy, comprising administering atherapeutically effective amount of the Factor VII polypeptide of claim1 to a subject in need thereof.